Lactobacillus buchneri genotyping on the basis of clustered regularly interspaced short palindromic repeat (CRISPR) locus diversity.

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Briner, Alexandra E; Barrangou, Rodolphe

2014-02-01

Clustered regularly interspaced short palindromic repeats (CRISPR) in combination with associated sequences (cas) constitute the CRISPR-Cas immune system, which uptakes DNA from invasive genetic elements as novel "spacers" that provide a genetic record of immunization events. We investigated the potential of CRISPR-based genotyping of Lactobacillus buchneri, a species relevant for commercial silage, bioethanol, and vegetable fermentations. Upon investigating the occurrence and diversity of CRISPR-Cas systems in Lactobacillus buchneri genomes, we observed a ubiquitous occurrence of CRISPR arrays containing a 36-nucleotide (nt) type II-A CRISPR locus adjacent to four cas genes, including the universal cas1 and cas2 genes and the type II signature gene cas9. Comparative analysis of CRISPR spacer content in 26 L. buchneri pickle fermentation isolates associated with spoilage revealed 10 unique locus genotypes that contained between 9 and 29 variable spacers. We observed a set of conserved spacers at the ancestral end, reflecting a common origin, as well as leader-end polymorphisms, reflecting recent divergence. Some of these spacers showed perfect identity with phage sequences, and many spacers showed homology to Lactobacillus plasmid sequences. Following a comparative analysis of sequences immediately flanking protospacers that matched CRISPR spacers, we identified a novel putative protospacer-adjacent motif (PAM), 5'-AAAA-3'. Overall, these findings suggest that type II-A CRISPR-Cas systems are valuable for genotyping of L. buchneri.

Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein.

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Nam, Ki Hyun; Ding, Fran; Haitjema, Charles; Huang, Qingqiu; DeLisa, Matthew P; Ke, Ailong

2012-10-19

The CRISPR (clustered regularly interspaced short palindromic repeats) system is a prokaryotic RNA-based adaptive immune system against extrachromosomal genetic elements. Cas2 is a universally conserved core CRISPR-associated protein required for the acquisition of new spacers for CRISPR adaptation. It was previously characterized as an endoribonuclease with preference for single-stranded (ss)RNA. Here, we show using crystallography, mutagenesis, and isothermal titration calorimetry that the Bacillus halodurans Cas2 (Bha_Cas2) from the subtype I-C/Dvulg CRISPR instead possesses metal-dependent endonuclease activity against double-stranded (ds)DNA. This activity is consistent with its putative function in producing new spacers for insertion into the 5'-end of the CRISPR locus. Mutagenesis and isothermal titration calorimetry studies revealed that a single divalent metal ion (Mg(2+) or Mn(2+)), coordinated by a symmetric Asp pair in the Bha_Cas2 dimer, is involved in the catalysis. We envision that a pH-dependent conformational change switches Cas2 into a metal-binding competent conformation for catalysis. We further propose that the distinct substrate preferences among Cas2 proteins may be determined by the sequence and structure in the β1-α1 loop.

Molecular identification and characterization of clustered regularly interspaced short palindromic repeat (CRISPR) gene cluster in Taylorella equigenitalis.

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Hara, Yasushi; Hayashi, Kyohei; Nakajima, Takuya; Kagawa, Shizuko; Tazumi, Akihiro; Moore, John E; Matsuda, Motoo

2013-09-01

Clustered regularly interspaced short palindromic repeats (CRISPRs), of approximately 10,000 base pairs (bp) in length, were shown to occur in the Japanese Taylorella equigenitalis strain, EQ59. The locus was composed of the putative CRISPRs-associated with 5 (cas5), RAMP csd1, csd2, recB, cas1, a leader region, 13 CRISPR consensus sequence repeats (each 32 bp; 5'-TCAGCCACGTTCGCGTGGCTGTGTGTTTAAAG-3'). These were in turn separated by 12 non repetitive unique spacer regions of similar length. In addition, a leader region, a transposase/IS protein, a leader region, and cas3 were also seen. All seven putative open reading frames carry their ribosome binding sites. Promoter consensus sequences at the -35 and -10 regions and putative intrinsic ρ-independent transcription terminator regions also occurred. A possible long overlap of 170 bp in length occurred between the recB and cas1 loci. Positive reverse transcription PCR signals of cas5, RAMP csd1, csd2-recB/cas1, and cas3 were generated. A putative secondary structure of the CRISPR consensus repeats was constructed. Following this, CRISPR results of the T. equigenitalis EQ59 isolate were subsequently compared with those from the Taylorella asinigenitalis MCE3 isolate.

Gene Repression in Haloarchaea Using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas I-B System.

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Stachler, Aris-Edda; Marchfelder, Anita

2016-07-15

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is used by bacteria and archaea to fend off foreign genetic elements. Since its discovery it has been developed into numerous applications like genome editing and regulation of transcription in eukaryotes and bacteria. For archaea currently no tools for transcriptional repression exist. Because molecular biology analyses in archaea become more and more widespread such a tool is vital for investigating the biological function of essential genes in archaea. Here we use the model archaeon Haloferax volcanii to demonstrate that its endogenous CRISPR-Cas system I-B can be harnessed to repress gene expression in archaea. Deletion of cas3 and cas6b genes results in efficient repression of transcription. crRNAs targeting the promoter region reduced transcript levels down to 8%. crRNAs targeting the reading frame have only slight impact on transcription. crRNAs that target the coding strand repress expression only down to 88%, whereas crRNAs targeting the template strand repress expression down to 8%. Repression of an essential gene results in reduction of transcription levels down to 22%. Targeting efficiencies can be enhanced by expressing a catalytically inactive Cas3 mutant. Genes can be targeted on plasmids or on the chromosome, they can be monocistronic or part of a polycistronic operon. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Gene Repression in Haloarchaea Using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas I-B System*

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Stachler, Aris-Edda; Marchfelder, Anita

2016-01-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is used by bacteria and archaea to fend off foreign genetic elements. Since its discovery it has been developed into numerous applications like genome editing and regulation of transcription in eukaryotes and bacteria. For archaea currently no tools for transcriptional repression exist. Because molecular biology analyses in archaea become more and more widespread such a tool is vital for investigating the biological function of essential genes in archaea. Here we use the model archaeon Haloferax volcanii to demonstrate that its endogenous CRISPR-Cas system I-B can be harnessed to repress gene expression in archaea. Deletion of cas3 and cas6b genes results in efficient repression of transcription. crRNAs targeting the promoter region reduced transcript levels down to 8%. crRNAs targeting the reading frame have only slight impact on transcription. crRNAs that target the coding strand repress expression only down to 88%, whereas crRNAs targeting the template strand repress expression down to 8%. Repression of an essential gene results in reduction of transcription levels down to 22%. Targeting efficiencies can be enhanced by expressing a catalytically inactive Cas3 mutant. Genes can be targeted on plasmids or on the chromosome, they can be monocistronic or part of a polycistronic operon. PMID:27226589

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) RNAs in the Porphyromonas gingivalis CRISPR-Cas I-C System.

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Burmistrz, Michal; Rodriguez Martinez, Jose Ignacio; Krochmal, Daniel; Staniec, Dominika; Pyrc, Krzysztof

2017-12-01

The CRISPR-Cas (clustered regularly interspaced short palindromic repeat-CRISPR-associated protein) system is unique to prokaryotes and provides the majority of bacteria and archaea with immunity against nucleic acids of foreign origin. CRISPR RNAs (crRNAs) are the key element of this system, since they are responsible for its selectivity and effectiveness. Typical crRNAs consist of a spacer sequence flanked with 5' and 3' handles originating from repeat sequences that are important for recognition of these small RNAs by the Cas machinery. In this investigation, we studied the type I-C CRISPR-Cas system in Porphyromonas gingivalis , a human pathogen associated with periodontitis, rheumatoid arthritis, cardiovascular disease, and aspiration pneumonia. We demonstrated the importance of the 5' handle for crRNA recognition by the effector complex and consequently activity, as well as secondary trimming of the 3' handle, which was not affected by modifications of the repeat sequence. IMPORTANCE Porphyromonas gingivalis , a clinically relevant Gram-negative, anaerobic bacterium, is one of the major etiologic agents of periodontitis and has been linked with the development of other clinical conditions, including rheumatoid arthritis, cardiovascular disease, and aspiration pneumonia. The presented results on the biogenesis and functions of crRNAs expand our understanding of CRISPR-Cas cellular defenses in P. gingivalis and of horizontal gene transfer in bacteria. Copyright © 2017 American Society for Microbiology.

Characterization of Genomic Deletion Efficiency Mediated by Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 Nuclease System in Mammalian Cells*♦

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Canver, Matthew C.; Bauer, Daniel E.; Dass, Abhishek; Yien, Yvette Y.; Chung, Jacky; Masuda, Takeshi; Maeda, Takahiro; Paw, Barry H.; Orkin, Stuart H.

2014-01-01

The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements. PMID:24907273

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 with improved proof-reading enhances homology-directed repair.

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Kato-Inui, Tomoko; Takahashi, Gou; Hsu, Szuyin; Miyaoka, Yuichiro

2018-05-18

Genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) predominantly induces non-homologous end joining (NHEJ), which generates random insertions or deletions, whereas homology-directed repair (HDR), which generates precise recombination products, is useful for wider applications. However, the factors that determine the ratio of HDR to NHEJ products after CRISPR/Cas9 editing remain unclear, and methods by which the proportion of HDR products can be increased have not yet been fully established. We systematically analyzed the HDR and NHEJ products after genome editing using various modified guide RNAs (gRNAs) and Cas9 variants with an enhanced conformational checkpoint to improve the fidelity at endogenous gene loci in HEK293T cells and HeLa cells. We found that these modified gRNAs and Cas9 variants were able to enhance HDR in both single-nucleotide substitutions and a multi-kb DNA fragment insertion. Our results suggest that the original CRISPR/Cas9 system from the bacterial immune system is not necessarily the best option for the induction of HDR in genome editing and indicate that the modulation of the kinetics of conformational checkpoints of Cas9 can optimize the HDR/NHEJ ratio.

Clustered, regularly interspaced short palindromic repeat (CRISPR) diversity and virulence factor distribution in avian Escherichia coli.

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Fu, Qiang; Su, Zhixin; Cheng, Yuqiang; Wang, Zhaofei; Li, Shiyu; Wang, Heng'an; Sun, Jianhe; Yan, Yaxian

In order to investigate the diverse characteristics of clustered, regularly interspaced short palindromic repeat (CRISPR) arrays and the distribution of virulence factor genes in avian Escherichia coli, 80 E. coli isolates obtained from chickens with avian pathogenic E. coli (APEC) or avian fecal commensal E. coli (AFEC) were identified. Using the multiplex polymerase chain reaction (PCR), five genes were subjected to phylogenetic typing and examined for CRISPR arrays to study genetic relatedness among the strains. The strains were further analyzed for CRISPR loci and virulence factor genes to determine a possible association between their CRISPR elements and their potential virulence. The strains were divided into five phylogenetic groups: A, B1, B2, D and E. It was confirmed that two types of CRISPR arrays, CRISPR1 and CRISPR2, which contain up to 246 distinct spacers, were amplified in most of the strains. Further classification of the isolates was achieved by sorting them into nine CRISPR clusters based on their spacer profiles, which indicates a candidate typing method for E. coli. Several significant differences in invasion-associated gene distribution were found between the APEC isolates and the AFEC isolates. Our results identified the distribution of 11 virulence genes and CRISPR diversity in 80 strains. It was demonstrated that, with the exception of iucD and aslA, there was no sharp demarcation in the gene distribution between the pathogenic (APEC) and commensal (AFEC) strains, while the total number of indicated CRISPR spacers may have a positive correlation with the potential pathogenicity of the E. coli isolates. Copyright © 2016. Published by Elsevier Masson SAS.

Characterization of genomic deletion efficiency mediated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells.

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Canver, Matthew C; Bauer, Daniel E; Dass, Abhishek; Yien, Yvette Y; Chung, Jacky; Masuda, Takeshi; Maeda, Takahiro; Paw, Barry H; Orkin, Stuart H

2014-08-01

The clustered regularly interspaced short [corrected] palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

High-temperature protein G is essential for activity of the Escherichia coli clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system.

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Yosef, Ido; Goren, Moran G; Kiro, Ruth; Edgar, Rotem; Qimron, Udi

2011-12-13

Prokaryotic DNA arrays arranged as clustered regularly interspaced short palindromic repeats (CRISPR), along with their associated proteins, provide prokaryotes with adaptive immunity by RNA-mediated targeting of alien DNA or RNA matching the sequences between the repeats. Here, we present a thorough screening system for the identification of bacterial proteins participating in immunity conferred by the Escherichia coli CRISPR system. We describe the identification of one such protein, high-temperature protein G (HtpG), a homolog of the eukaryotic chaperone heat-shock protein 90. We demonstrate that in the absence of htpG, the E. coli CRISPR system loses its suicidal activity against λ prophage and its ability to provide immunity from lysogenization. Transcomplementation of htpG restores CRISPR activity. We further show that inactivity of the CRISPR system attributable to htpG deficiency can be suppressed by expression of Cas3, a protein that is essential for its activity. Accordingly, we also find that the steady-state level of overexpressed Cas3 is significantly enhanced following HtpG expression. We conclude that HtpG is a newly identified positive modulator of the CRISPR system that is essential for maintaining functional levels of Cas3.

Molecular identification and characterization of clustered regularly interspaced short palindromic repeats (CRISPRs) in a urease-positive thermophilic Campylobacter sp. (UPTC).

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Tasaki, E; Hirayama, J; Tazumi, A; Hayashi, K; Hara, Y; Ueno, H; Moore, J E; Millar, B C; Matsuda, M

2012-02-01

Novel clustered regularly-interspaced short palindromic repeats (CRISPRs) locus [7,500 base pairs (bp) in length] occurred in the urease-positive thermophilic Campylobacter (UPTC) Japanese isolate, CF89-12. The 7,500 bp gene loci consisted of the 5'-methylaminomethyl-2-thiouridylate methyltransferase gene, putative (P) CRISPR associated (p-Cas), putative open reading frames, Cas1 and Cas2, leader sequence region (146 bp), 12 CRISPRs consensus sequence repeats (each 36 bp) separated by a non-repetitive unique spacer region of similar length (26-31 bp) and the phosphatidyl glycerophosphatase A gene. When the CRISPRs loci in the UPTC CF89-12 and five C. jejuni isolates were compared with one another, these six isolates contained p-Cas, Cas1 and Cas2 within the loci. Four to 12 CRISPRs consensus sequence repeats separated by a non-repetitive unique spacer region occurred in six isolates and the nucleotide sequences of those repeats gave approximately 92-100% similarity with each other. However, no sequence similarity occurred in the unique spacer regions among these isolates. The putative σ(70) transcriptional promoter and the hypothetical ρ-independent terminator structures for the CRISPRs and Cas were detected. No in vivo transcription of p-Cas, Cas1 and Cas2 was confirmed in the UPTC cells.

Transgenic Clustered Regularly Interspaced Short Palindromic Repeat/Cas9-Mediated Viral Gene Targeting for Antiviral Therapy of Bombyx mori Nucleopolyhedrovirus.

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Chen, Shuqing; Hou, Chengxiang; Bi, Honglun; Wang, Yueqiang; Xu, Jun; Li, Muwang; James, Anthony A; Huang, Yongping; Tan, Anjiang

2017-04-15

We developed a novel antiviral strategy by combining transposon-based transgenesis and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system for the direct cleavage of Bombyx mori nucleopolyhedrovirus (BmNPV) genome DNA to promote virus clearance in silkworms. We demonstrate that transgenic silkworms constitutively expressing Cas9 and guide RNAs targeting the BmNPV immediate early-1 ( ie-1 ) and me53 genes effectively induce target-specific cleavage and subsequent mutagenesis, especially large (∼7-kbp) segment deletions in BmNPV genomes, and thus exhibit robust suppression of BmNPV proliferation. Transgenic animals exhibited higher and inheritable resistance to BmNPV infection than wild-type animals. Our approach will not only contribute to modern sericulture but also shed light on future antiviral therapy. IMPORTANCE Pathogen genome targeting has shown its potential in antiviral research. However, transgenic CRISPR/Cas9 system-mediated viral genome targeting has not been reported as an antiviral strategy in a natural animal host of a virus. Our data provide an effective approach against BmNPV infection in a real-world biological system and demonstrate the potential of transgenic CRISPR/Cas9 systems in antiviral research in other species. Copyright © 2017 Chen et al.

Comparisons of clustered regularly interspaced short palindromic repeats and viromes in human saliva reveal bacterial adaptations to salivary viruses.

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Pride, David T; Salzman, Julia; Relman, David A

2012-09-01

Explorations of human microbiota have provided substantial insight into microbial community composition; however, little is known about interactions between various microbial components in human ecosystems. In response to the powerful impact of viral predation, bacteria have acquired potent defences, including an adaptive immune response based on the clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas system. To improve our understanding of the interactions between bacteria and their viruses in humans, we analysed 13 977 streptococcal CRISPR sequences and compared them with 2 588 172 virome reads in the saliva of four human subjects over 17 months. We found a diverse array of viruses and CRISPR spacers, many of which were specific to each subject and time point. There were numerous viral sequences matching CRISPR spacers; these matches were highly specific for salivary viruses. We determined that spacers and viruses coexist at the same time, which suggests that streptococcal CRISPR/Cas systems are under constant pressure from salivary viruses. CRISPRs in some subjects were just as likely to match viral sequences from other subjects as they were to match viruses from the same subject. Because interactions between bacteria and viruses help to determine the structure of bacterial communities, CRISPR-virus analyses are likely to provide insight into the forces shaping the human microbiome. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site.

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Hatoum-Aslan, Asma; Maniv, Inbal; Marraffini, Luciano A

2011-12-27

Precise RNA processing is fundamental to all small RNA-mediated interference pathways. In prokaryotes, clustered, regularly interspaced, short palindromic repeats (CRISPR) loci encode small CRISPR RNAs (crRNAs) that protect against invasive genetic elements by antisense targeting. CRISPR loci are transcribed as a long precursor that is cleaved within repeat sequences by CRISPR-associated (Cas) proteins. In many organisms, this primary processing generates crRNA intermediates that are subject to additional nucleolytic trimming to render mature crRNAs of specific lengths. The molecular mechanisms underlying this maturation event remain poorly understood. Here, we defined the genetic requirements for crRNA primary processing and maturation in Staphylococcus epidermidis. We show that changes in the position of the primary processing site result in extended or diminished maturation to generate mature crRNAs of constant length. These results indicate that crRNA maturation occurs by a ruler mechanism anchored at the primary processing site. We also show that maturation is mediated by specific cas genes distinct from those genes involved in primary processing, showing that this event is directed by CRISPR/Cas loci.

[Knocking-out extra domain A alternative splice fragment of fibronectin using a clustered regularly interspaced short palindromic repeats/associated proteins 9 system].

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Yang, Yue; Wang, Haicheng; Xu, Shuyu; Peng, Jing; Jiang, Jiuhui; Li, Cuiying

2015-08-01

To investigate the effect of the fibronectin extra domain A on the aggressiveness of salivary adenoid cystic carcinoma (SACC) cells, via the clustered regularly interspaced short palindromic repeats (CRISPR)/ associated proteins (Cas) system. One sgRNA was designed to target the upstream of the genome sequences of extra domain A(EDA) exon and the downstream. Then the sgRNA was linked into plasmid PX-330 and transfected into SACC-83 cells. PCR and DNA sequence were used to testify the knockout cells, and the monoclones of EDA absent SACC cells were selected (A+C-2, A+C-6, B+C-10). CCK-8 cell proliferation and invasion was then tested in control group and the experimental group. The sgRNA was successfully linked into PX-330 plasmid. Part of adenoid cystic carcinoma cells' SACC-83 genomic EDA exon was knocked out, and the knockdown efficiency was above 70%, but the total amount of fibronectin did not change significantly. Three monoclones of EDA absent SACC- 83 cells were successfully selected with diminished migration and proliferation. The CRISPR/Cas9 system was a simplified system with relatively high knockout efficiency and EDA knockout could inhibiting SACC cell's mobility and invasiveness.

Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic repeat (CRISPR)-associated complex for antiviral defense (CASCADE).

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Lintner, Nathanael G; Kerou, Melina; Brumfield, Susan K; Graham, Shirley; Liu, Huanting; Naismith, James H; Sdano, Matthew; Peng, Nan; She, Qunxin; Copié, Valérie; Young, Mark J; White, Malcolm F; Lawrence, C Martin

2011-06-17

In response to viral infection, many prokaryotes incorporate fragments of virus-derived DNA into loci called clustered regularly interspaced short palindromic repeats (CRISPRs). The loci are then transcribed, and the processed CRISPR transcripts are used to target invading viral DNA and RNA. The Escherichia coli "CRISPR-associated complex for antiviral defense" (CASCADE) is central in targeting invading DNA. Here we report the structural and functional characterization of an archaeal CASCADE (aCASCADE) from Sulfolobus solfataricus. Tagged Csa2 (Cas7) expressed in S. solfataricus co-purifies with Cas5a-, Cas6-, Csa5-, and Cas6-processed CRISPR-RNA (crRNA). Csa2, the dominant protein in aCASCADE, forms a stable complex with Cas5a. Transmission electron microscopy reveals a helical complex of variable length, perhaps due to substoichiometric amounts of other CASCADE components. A recombinant Csa2-Cas5a complex is sufficient to bind crRNA and complementary ssDNA. The structure of Csa2 reveals a crescent-shaped structure unexpectedly composed of a modified RNA-recognition motif and two additional domains present as insertions in the RNA-recognition motif. Conserved residues indicate potential crRNA- and target DNA-binding sites, and the H160A variant shows significantly reduced affinity for crRNA. We propose a general subunit architecture for CASCADE in other bacteria and Archaea.

An Active Immune Defense with a Minimal CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA and without the Cas6 Protein*

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Maier, Lisa-Katharina; Stachler, Aris-Edda; Saunders, Sita J.; Backofen, Rolf; Marchfelder, Anita

2015-01-01

The prokaryotic immune system CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) is a defense system that protects prokaryotes against foreign DNA. The short CRISPR RNAs (crRNAs) are central components of this immune system. In CRISPR-Cas systems type I and III, crRNAs are generated by the endonuclease Cas6. We developed a Cas6b-independent crRNA maturation pathway for the Haloferax type I-B system in vivo that expresses a functional crRNA, which we termed independently generated crRNA (icrRNA). The icrRNA is effective in triggering degradation of an invader plasmid carrying the matching protospacer sequence. The Cas6b-independent maturation of the icrRNA allowed mutation of the repeat sequence without interfering with signals important for Cas6b processing. We generated 23 variants of the icrRNA and analyzed them for activity in the interference reaction. icrRNAs with deletions or mutations of the 3′ handle are still active in triggering an interference reaction. The complete 3′ handle could be removed without loss of activity. However, manipulations of the 5′ handle mostly led to loss of interference activity. Furthermore, we could show that in the presence of an icrRNA a strain without Cas6b (Δcas6b) is still active in interference. PMID:25512373

An active immune defense with a minimal CRISPR (clustered regularly interspaced short palindromic repeats) RNA and without the Cas6 protein.

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Maier, Lisa-Katharina; Stachler, Aris-Edda; Saunders, Sita J; Backofen, Rolf; Marchfelder, Anita

2015-02-13

The prokaryotic immune system CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) is a defense system that protects prokaryotes against foreign DNA. The short CRISPR RNAs (crRNAs) are central components of this immune system. In CRISPR-Cas systems type I and III, crRNAs are generated by the endonuclease Cas6. We developed a Cas6b-independent crRNA maturation pathway for the Haloferax type I-B system in vivo that expresses a functional crRNA, which we termed independently generated crRNA (icrRNA). The icrRNA is effective in triggering degradation of an invader plasmid carrying the matching protospacer sequence. The Cas6b-independent maturation of the icrRNA allowed mutation of the repeat sequence without interfering with signals important for Cas6b processing. We generated 23 variants of the icrRNA and analyzed them for activity in the interference reaction. icrRNAs with deletions or mutations of the 3' handle are still active in triggering an interference reaction. The complete 3' handle could be removed without loss of activity. However, manipulations of the 5' handle mostly led to loss of interference activity. Furthermore, we could show that in the presence of an icrRNA a strain without Cas6b (Δcas6b) is still active in interference. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Diversity, evolution, and functionality of clustered regularly interspaced short palindromic repeat (CRISPR) regions in the fire blight pathogen Erwinia amylovora.

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Rezzonico, Fabio; Smits, Theo H M; Duffy, Brion

2011-06-01

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system confers acquired heritable immunity against mobile nucleic acid elements in prokaryotes, limiting phage infection and horizontal gene transfer of plasmids. In CRISPR arrays, characteristic repeats are interspersed with similarly sized nonrepetitive spacers derived from transmissible genetic elements and acquired when the cell is challenged with foreign DNA. New spacers are added sequentially and the number and type of CRISPR units can differ among strains, providing a record of phage/plasmid exposure within a species and giving a valuable typing tool. The aim of this work was to investigate CRISPR diversity in the highly homogeneous species Erwinia amylovora, the causal agent of fire blight. A total of 18 CRISPR genotypes were defined within a collection of 37 cosmopolitan strains. Strains from Spiraeoideae plants clustered in three major groups: groups II and III were composed exclusively of bacteria originating from the United States, whereas group I generally contained strains of more recent dissemination obtained in Europe, New Zealand, and the Middle East. Strains from Rosoideae and Indian hawthorn (Rhaphiolepis indica) clustered separately and displayed a higher intrinsic diversity than that of isolates from Spiraeoideae plants. Reciprocal exclusion was generally observed between plasmid content and cognate spacer sequences, supporting the role of the CRISPR/Cas system in protecting against foreign DNA elements. However, in several group III strains, retention of plasmid pEU30 is inconsistent with a functional CRISPR/Cas system.

Association of clustered regularly interspaced short palindromic repeat (CRISPR) elements with specific serotypes and virulence potential of shiga toxin-producing Escherichia coli.

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Toro, Magaly; Cao, Guojie; Ju, Wenting; Allard, Marc; Barrangou, Rodolphe; Zhao, Shaohua; Brown, Eric; Meng, Jianghong

2014-02-01

Shiga toxin-producing Escherichia coli (STEC) strains (n = 194) representing 43 serotypes and E. coli K-12 were examined for clustered regularly interspaced short palindromic repeat (CRISPR) arrays to study genetic relatedness among STEC serotypes. A subset of the strains (n = 81) was further analyzed for subtype I-E cas and virulence genes to determine a possible association of CRISPR elements with potential virulence. Four types of CRISPR arrays were identified. CRISPR1 and CRISPR2 were present in all strains tested; 1 strain also had both CRISPR3 and CRISPR4, whereas 193 strains displayed a short, combined array, CRISPR3-4. A total of 3,353 spacers were identified, representing 528 distinct spacers. The average length of a spacer was 32 bp. Approximately one-half of the spacers (54%) were unique and found mostly in strains of less common serotypes. Overall, CRISPR spacer contents correlated well with STEC serotypes, and identical arrays were shared between strains with the same H type (O26:H11, O103:H11, and O111:H11). There was no association identified between the presence of subtype I-E cas and virulence genes, but the total number of spacers had a negative correlation with potential pathogenicity (P CRISPR-cas system and potential virulence needs to be determined on a broader scale, and the biological link will need to be established.

Characterization of the clustered regularly interspaced short palindromic repeats sites in Streptococcus mutans isolated from early childhood caries patients.

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Chen, Jing; Li, Tiancheng; Zhou, Xuedong; Cheng, Lei; Huo, Yuanyuan; Zou, Jing; Li, Yuqing

2017-11-01

The aim of this study was to analyze the characteristics of the clustered regularly interspaced short palindromic repeats (CRISPR) sites in 45 clinical Streptococcus mutans strains and their relationship to the clinical manifestations of early childhood caries (ECC). Forty-five S. mutans strains were isolated from the plaque samples taken from sixty-three children. CRISPR sites were sequenced and BLAST was used to compare these sites to those in the CRISPRTarget database. The association between the distribution of CRISPR sites and the manifestation of caries was analyzed by Chi-Square test. Further, biofilm formation (by crystal violet staining) and the synthesis of polysaccharide (by anthrone-sulfuric method) of all clinical isolated S. mutans strains with both CRISPR sites and no CRISPR site were comapared. Finally, acidogenicity and acidurity of two typical strains were determined using pH drop and acid tolerance assays. Biofilm formation and EPS synthesis by two typical strains were compared by 3D CLSM (Confocal Laser Scanning Microscope) assays and the expression of gtf genes were evaluated using qPCR. We found that most of the spacers in the clinical S. mutans strains were derived from Streptococcus phages APCM01 and M102. The number of CRISPR sites in these strains was associated with the clinical manifestations of ECC. Moreover, we found that the biofilm formation and EPS synthesis ability of the S. mutans strains with both CRISPR sites was significant improved. An association was found between the distribution of CRISPR sites and the clinical manifestations of caries. The CRISPR sites might contribute to the cariogenic potential of S. mutans. Copyright © 2017 Elsevier Ltd. All rights reserved.

Crystal structure of clustered regularly interspaced short palindromic repeats (CRISPR)-associated Csn2 protein revealed Ca2+-dependent double-stranded DNA binding activity.

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Nam, Ki Hyun; Kurinov, Igor; Ke, Ailong

2011-09-02

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 Å tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is ∼26 Å wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an α/β domain and an α-helical domain; significant hinge motion was observed between these two domains. Ca(2+) was located at strategic positions in the oligomerization interface. We further showed that removal of Ca(2+) ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca(2+) ions.

Subtyping Salmonella enterica serovar enteritidis isolates from different sources by using sequence typing based on virulence genes and clustered regularly interspaced short palindromic repeats (CRISPRs).

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Liu, Fenyun; Kariyawasam, Subhashinie; Jayarao, Bhushan M; Barrangou, Rodolphe; Gerner-Smidt, Peter; Ribot, Efrain M; Knabel, Stephen J; Dudley, Edward G

2011-07-01

Salmonella enterica subsp. enterica serovar Enteritidis is a major cause of food-borne salmonellosis in the United States. Two major food vehicles for S. Enteritidis are contaminated eggs and chicken meat. Improved subtyping methods are needed to accurately track specific strains of S. Enteritidis related to human salmonellosis throughout the chicken and egg food system. A sequence typing scheme based on virulence genes (fimH and sseL) and clustered regularly interspaced short palindromic repeats (CRISPRs)-CRISPR-including multi-virulence-locus sequence typing (designated CRISPR-MVLST)-was used to characterize 35 human clinical isolates, 46 chicken isolates, 24 egg isolates, and 63 hen house environment isolates of S. Enteritidis. A total of 27 sequence types (STs) were identified among the 167 isolates. CRISPR-MVLST identified three persistent and predominate STs circulating among U.S. human clinical isolates and chicken, egg, and hen house environmental isolates in Pennsylvania, and an ST that was found only in eggs and humans. It also identified a potential environment-specific sequence type. Moreover, cluster analysis based on fimH and sseL identified a number of clusters, of which several were found in more than one outbreak, as well as 11 singletons. Further research is needed to determine if CRISPR-MVLST might help identify the ecological origins of S. Enteritidis strains that contaminate chickens and eggs.

Crystal Structure of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Csn2 Protein Revealed Ca[superscript 2+]-dependent Double-stranded DNA Binding Activity

Energy Technology Data Exchange (ETDEWEB)

Nam, Ki Hyun; Kurinov, Igor; Ke, Ailong (Cornell); (NWU)

2012-05-22

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 {angstrom} tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is {approx}26 {angstrom} wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an {alpha}/{beta} domain and an {alpha}-helical domain; significant hinge motion was observed between these two domains. Ca{sup 2+} was located at strategic positions in the oligomerization interface. We further showed that removal of Ca{sup 2+} ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca{sup 2+} ions.

Intricate interactions between the bloom-forming cyanobacterium Microcystis aeruginosa and foreign genetic elements, revealed by diversified clustered regularly interspaced short palindromic repeat (CRISPR) signatures.

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Kuno, Sotaro; Yoshida, Takashi; Kaneko, Takakazu; Sako, Yoshihiko

2012-08-01

Clustered regularly interspaced short palindromic repeats (CRISPR) confer sequence-dependent, adaptive resistance in prokaryotes against viruses and plasmids via incorporation of short sequences, called spacers, derived from foreign genetic elements. CRISPR loci are thus considered to provide records of past infections. To describe the host-parasite (i.e., cyanophages and plasmids) interactions involving the bloom-forming freshwater cyanobacterium Microcystis aeruginosa, we investigated CRISPR in four M. aeruginosa strains and in two previously sequenced genomes. The number of spacers in each locus was larger than the average among prokaryotes. All spacers were strain specific, except for a string of 11 spacers shared in two closely related strains, suggesting diversification of the loci. Using CRISPR repeat-based PCR, 24 CRISPR genotypes were identified in a natural cyanobacterial community. Among 995 unique spacers obtained, only 10 sequences showed similarity to M. aeruginosa phage Ma-LMM01. Of these, six spacers showed only silent or conservative nucleotide mutations compared to Ma-LMM01 sequences, suggesting a strategy by the cyanophage to avert CRISPR immunity dependent on nucleotide identity. These results imply that host-phage interactions can be divided into M. aeruginosa-cyanophage combinations rather than pandemics of population-wide infectious cyanophages. Spacer similarity also showed frequent exposure of M. aeruginosa to small cryptic plasmids that were observed only in a few strains. Thus, the diversification of CRISPR implies that M. aeruginosa has been challenged by diverse communities (almost entirely uncharacterized) of cyanophages and plasmids.

The evolutionary divergence of Shiga toxin-producing Escherichia coli is reflected in clustered regularly interspaced short palindromic repeat (CRISPR) spacer composition.

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Yin, Shuang; Jensen, Mark A; Bai, Jiawei; Debroy, Chitrita; Barrangou, Rodolphe; Dudley, Edward G

2013-09-01

The Shiga toxin-producing Escherichia coli (STEC) strains, including those of O157:H7 and the "big six" serogroups (i.e., serogroups O26, O45, O103, O111, O121, and O145), are a group of pathogens designated food adulterants in the United States. The relatively conserved nature of clustered regularly interspaced short palindromic repeats (CRISPRs) in phylogenetically related E. coli strains makes them potential subtyping markers for STEC detection, and a quantitative PCR (qPCR)-based assay was previously developed for O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7 isolates. To better evaluate the sensitivity and specificity of this qPCR method, the CRISPR loci of 252 O157 and big-six STEC isolates were sequenced and analyzed along with 563 CRISPR1 and 624 CRISPR2 sequences available in GenBank. General conservation of spacer content and order was observed within each O157 and big-six serogroup, validating the qPCR method. Meanwhile, it was found that spacer deletion, the presence of an insertion sequence, and distinct alleles within a serogroup are sources of false-negative reactions. Conservation of CRISPR arrays among isolates expressing the same flagellar antigen, specifically, H7, H2, and H11, suggested that these isolates share an ancestor and provided an explanation for the false positives previously observed in the qPCR results. An analysis of spacer distribution across E. coli strains provided limited evidence for temporal spacer acquisition. Conversely, comparison of CRISPR sequences between strains along the stepwise evolution of O157:H7 from its O55:H7 ancestor revealed that, over this ∼7,000-year span, spacer deletion was the primary force generating CRISPR diversity.

Generation of Hypertension-Associated STK39 Polymorphism Knockin Cell Lines With the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 System.

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Mandai, Shintaro; Mori, Takayasu; Sohara, Eisei; Rai, Tatemitsu; Uchida, Shinichi

2015-12-01

Previous genome-wide association studies identified serine threonine kinase 39 (STK39), encoding STE20/SPS1-related proline/alanine-rich kinase, as one of a limited number of hypertension susceptibility genes. A recent meta-analysis confirmed the association of STK39 intronic polymorphism rs3754777 with essential hypertension, among previously reported hypertension-associated STK39 polymorphisms. However, the biochemical function of this polymorphism in the mechanism responsible for hypertension is yet to be clarified. We generated rs3754777G>A knockin human cell lines with clustered regularly interspaced short palindromic repeats-mediated genome engineering. Homozygous (A/A) and heterozygous (G/A) knockin human embryonic kidney cell lines were generated using a double nickase, single-guide RNAs targeting STK39 intron 5 around single-nucleotide polymorphism, and a 100-bp donor single-stranded DNA oligonucleotide. Reverse transcription polymerase chain reaction with sequencing analyses revealed the identical STK39 transcripts among the wild-type and both knockin cell lines. Quantitative reverse transcription polymerase chain reaction showed increased STK39 mRNA expression, and immunoblot analysis revealed increases in total and phosphorylated STE20/SPS1-related proline/alanine-rich kinase with increased phosphorylated Na-K-Cl cotransporter isoform 1 in both knockin cell lines. The largest increases in these molecules were observed in the homozygous cell line. These findings indicated that this intronic polymorphism increases STK39 transcription, leading to activation of the STE20/SPS1-related proline/alanine-rich kinase-solute carrier family 12A signaling cascade. Increased interactions between STE20/SPS1-related proline/alanine-rich kinase and the target cation-chloride cotransporters may be responsible for hypertension susceptibility in individuals with this polymorphism. © 2015 American Heart Association, Inc.

Short communication: Determination of Salmonella clustered regularly interspaced short palindromic repeats (CRISPR) diversity on dairy farms in Wisconsin and Minnesota.

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Wehnes, C A; Rehberger, T G; Barrangou, R; Smith, A H

2014-10-01

Salmonella enterica ssp. enterica is a foodborne pathogen able to cause disease in both humans and animals. Diverse serovars of this pathogen exist, some of which are host specific, causing a range of clinical symptoms from asymptomatic infection through morbidity and mortality. According to a 2007 survey by the USDA National Animal Health Monitoring System, fecal shedding of Salmonella from healthy cows occurs on 39.7% of dairy farms in the United States. Certain serovars are frequently isolated from dairy farms and the majority of isolates from the National Animal Health Monitoring System study were represented by 5 serovars; however, genotypic diversity was not examined. The objective of this study was to determine the diversity of clustered regularly interspaced short palindromic repeats (CRISPR) loci in Salmonella collected from 8 dairy farms with a previous history of salmonellosis. None of the cows or calves sampled on 2 of the 8 dairy farms were shedding Salmonella, although Salmonella was detected in a cow bedding sample on 1 of these farms. Salmonella populations were discrete on each farm, according to CRISPR typing, with the exception of an Anatum var. 15+ type on farms 5 and 6 and the Montevideo type on farms 1 and 2. One to 4 distinct CRISPR genotypes were identified per farm. The CRISPR typing differed within serovars, as Montevideo, Anatum var. 15+, and Muenster serovars had no overlap of spacer content, even on the same farm, reflecting between- and within-serovar genetic diversity. The dynamic nature of Salmonella populations was shown in a farm that was sampled longitudinally over 13.5 mo. Changes in serovar from 3,19:-:z27 to Montevideo was observed between the first sampling time and 8 mo later, with concomitant change in CRISPR alleles. The results indicate that Salmonella strains present in smaller dairy herds (<500 head) are specific to that farm and new Salmonella strains may emerge over time. Copyright © 2014 American Dairy Science

Combination of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 technique with the piggybac transposon system for mouse in utero electroporation to study cortical development.

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Cheng, Man; Jin, Xubin; Mu, Lili; Wang, Fangyu; Li, Wei; Zhong, Xiaoling; Liu, Xuan; Shen, Wenchen; Liu, Ying; Zhou, Yan

2016-09-01

In utero electroporation (IUE) is commonly used to study cortical development of cerebrum by downregulating or overexpressing genes of interest in neural progenitor cells (NPCs) of small mammals. However, exogenous plasmids are lost or diluted over time. Furthermore, gene knockdown based on short-hairpin RNAs may exert nonspecific effects that lead to aberrant neuronal migration. Genomic engineering by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has great research and therapeutic potentials. Here we integrate the CRISPR/Cas9 components into the piggyBac (PB) transposon system (the CRISPR/Cas9-PB toolkit) for cortical IUEs. The mouse Sry-related HMG box-2 (Sox2) gene was selected as the target for its application. Most transduced cortical NPCs were depleted of SOX2 protein as early as 3 days post-IUE, whereas expressions of SOX1 and PAX6 remained intact. Furthermore, both the WT Cas9 and the D10A nickase mutant Cas9n showed comparable knockout efficiency. Transduced cortical cells were purified with fluorescence-activated cell sorting, and effective gene editing at the Sox2 loci was confirmed. Thus, application of the CRISPR/Cas9-PB toolkit in IUE is a promising strategy to study gene functions in cortical NPCs and their progeny. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Relationship between drug resistance and the clustered, regularly interspaced, short, palindromic repeat-associated protein genes cas1 and cas2 in Shigella from giant panda dung.

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Ren, Lu; Deng, Lin-Hua; Zhang, Ri-Peng; Wang, Cheng-Dong; Li, De-Sheng; Xi, Li-Xin; Chen, Zhen-Rong; Yang, Rui; Huang, Jie; Zeng, Yang-Ru; Wu, Hong-Lin; Cao, San-Jie; Wu, Rui; Huang, Yong; Yan, Qi-Gui

2017-02-01

To detect drug resistance in Shigella obtained from the dung of the giant panda, explore the factors leading to drug resistance in Shigella, understand the characteristics of clustered, regularly interspaced, short, palindromic repeats (CRISPR), and assess the relationship between CRISPR and drug resistance. We collected fresh feces from 27 healthy giant pandas in the Giant Panda Conservation base (Wolong, China). We identified the strains of Shigella in the samples by using nucleotide sequence analysis. Further, the Kirby-Bauer paper method was used to determine drug sensitivity of the Shigella strains. CRISPR-associated protein genes cas1 and cas2 in Shigella were detected by polymerase chain reaction (PCR), and the PCR products were sequenced and compared. We isolated and identified 17 strains of Shigella from 27 samples, including 14 strains of Shigella flexneri, 2 strains of Shigella sonnei, and 1 strain of Shigella dysenteriae. Further, drug resistance to cefazolin, imipenem, and amoxicillin-clavulanic acid was identified as a serious problem, as multidrug-resistant strains were detected. Further, cas1 and cas2 showed different degrees of point mutations. The CRISPR system widely exists in Shigella and shares homology with that in Escherichia coli. The cas1 and cas 2 mutations contribute to the different levels of resistance. Point mutations at sites 3176455, 3176590, and 3176465 in cas1 (a); sites 3176989, 3176992, and 3176995 in cas1 (b); sites 3176156 and 3176236 in cas2 may affect the resistance of bacteria, cause emergence of multidrug resistance, and increase the types of drug resistance.

Abundant and diverse clustered regularly interspaced short palindromic repeat spacers in Clostridium difficile strains and prophages target multiple phage types within this pathogen.

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Hargreaves, Katherine R; Flores, Cesar O; Lawley, Trevor D; Clokie, Martha R J

2014-08-26

Clostridium difficile is an important human-pathogenic bacterium causing antibiotic-associated nosocomial infections worldwide. Mobile genetic elements and bacteriophages have helped shape C. difficile genome evolution. In many bacteria, phage infection may be controlled by a form of bacterial immunity called the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system. This uses acquired short nucleotide sequences (spacers) to target homologous sequences (protospacers) in phage genomes. C. difficile carries multiple CRISPR arrays, and in this paper we examine the relationships between the host- and phage-carried elements of the system. We detected multiple matches between spacers and regions in 31 C. difficile phage and prophage genomes. A subset of the spacers was located in prophage-carried CRISPR arrays. The CRISPR spacer profiles generated suggest that related phages would have similar host ranges. Furthermore, we show that C. difficile strains of the same ribotype could either have similar or divergent CRISPR contents. Both synonymous and nonsynonymous mutations in the protospacer sequences were identified, as well as differences in the protospacer adjacent motif (PAM), which could explain how phages escape this system. This paper illustrates how the distribution and diversity of CRISPR spacers in C. difficile, and its prophages, could modulate phage predation for this pathogen and impact upon its evolution and pathogenicity. Clostridium difficile is a significant bacterial human pathogen which undergoes continual genome evolution, resulting in the emergence of new virulent strains. Phages are major facilitators of genome evolution in other bacterial species, and we use sequence analysis-based approaches in order to examine whether the CRISPR/Cas system could control these interactions across divergent C. difficile strains. The presence of spacer sequences in prophages that are homologous to phage genomes raises an

Native tandem and ion mobility mass spectrometry highlight structural and modular similarities in clustered-regularly-interspaced shot-palindromic-repeats (CRISPR)-associated protein complexes from Escherichia coli and Pseudomonas aeruginosa.

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van Duijn, Esther; Barbu, Ioana M; Barendregt, Arjan; Jore, Matthijs M; Wiedenheft, Blake; Lundgren, Magnus; Westra, Edze R; Brouns, Stan J J; Doudna, Jennifer A; van der Oost, John; Heck, Albert J R

2012-11-01

The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) immune system of bacteria and archaea provides acquired resistance against viruses and plasmids, by a strategy analogous to RNA-interference. Key components of the defense system are ribonucleoprotein complexes, the composition of which appears highly variable in different CRISPR/Cas subtypes. Previous studies combined mass spectrometry, electron microscopy, and small angle x-ray scattering to demonstrate that the E. coli Cascade complex (405 kDa) and the P. aeruginosa Csy-complex (350 kDa) are similar in that they share a central spiral-shaped hexameric structure, flanked by associating proteins and one CRISPR RNA. Recently, a cryo-electron microscopy structure of Cascade revealed that the CRISPR RNA molecule resides in a groove of the hexameric backbone. For both complexes we here describe the use of native mass spectrometry in combination with ion mobility mass spectrometry to assign a stable core surrounded by more loosely associated modules. Via computational modeling subcomplex structures were proposed that relate to the experimental IMMS data. Despite the absence of obvious sequence homology between several subunits, detailed analysis of sub-complexes strongly suggests analogy between subunits of the two complexes. Probing the specific association of E. coli Cascade/crRNA to its complementary DNA target reveals a conformational change. All together these findings provide relevant new information about the potential assembly process of the two CRISPR-associated complexes.

Relevance of the clustered regularly interspaced short palindromic repeats of Enterococcus faecalis strains isolated from retreatment root canals on periapical lesions, resistance to irrigants and biofilms.

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Tong, Zhongchun; Du, Yu; Ling, Junqi; Huang, Lijia; Ma, Jinglei

2017-12-01

A high prevalence of Enterococcus faecalis ( E. faecalis ) is observed in teeth with root canal treatment failures. Clustered regularly interspaced short palindromic repeats (CRISPR) are widely distributed in prokaryotes that have adaptive immune systems against mobile elements, including pathogenic genes. The present study investigated the relevance of the CRISPR in E. faecalis strains isolated from retreated root canals on biofilms, periapical lesions and drug resistance. A total of 20 E. faecalis strains were extracted from the root canals of teeth referred for root canal retreatment. CRISPR-Cas loci were identified by two pairs of relevant primers and polymerase chain reaction. The susceptibility of the 20 isolated strains to intracanal irrigants was evaluated by 1- and 5-minute challenges with a mixture of a tetracycline isomer, an acid and a detergent (MTAD), 2% chlorhexidine (CHX) and 5.25% sodium hypochlorite (NaOCl). The microtiter plate assay and crystal violet staining were used to compare the biofilm formation of the E. faecalis isolate strains. Out of the 20 E. faecalis isolate strains, 5 strains that lacked CRISPR-cas determinants exhibited significant periapical lesions. Among the 15 strains containing CRISPR-cas determinants, 8 were isolated from root canals with inadequate fillings and 7 were isolated from root canals without any fillings. The five strains lacking CRISPR-cas loci were observed to be more resistant to MTAD and 2% CHX than the 15 strains that had CRISPR-cas loci. All of the strains exhibited the same susceptibility to 5.25% NaOCl. Furthermore, the 5 strains lacking CRISPR-cas determinants generated more biofilm than the other 15 strains. Thus, the results of the present study suggested that E. faecalis root canal isolates lacking CRISPR-cas exhibit higher resistance to intracanal irrigants, stronger biofilm formation and generate significant periapical lesions.

The Clustered, Regularly Interspaced, Short Palindromic Repeats-associated Endonuclease 9 (CRISPR/Cas9)-created MDM2 T309G Mutation Enhances Vitreous-induced Expression of MDM2 and Proliferation and Survival of Cells.

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Duan, Yajian; Ma, Gaoen; Huang, Xionggao; D'Amore, Patricia A; Zhang, Feng; Lei, Hetian

2016-07-29

The G309 allele of SNPs in the mouse double minute (MDM2) promoter locus is associated with a higher risk of cancer and proliferative vitreoretinopathy (PVR), but whether SNP G309 contributes to the pathogenesis of PVR is to date unknown. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas) 9 from Streptococcus pyogenes (SpCas9) can be harnessed to manipulate a single or multiple nucleotides in mammalian cells. Here we delivered SpCas9 and guide RNAs using dual adeno-associated virus-derived vectors to target the MDM2 genomic locus together with a homologous repair template for creating the mutation of MDM2 T309G in human primary retinal pigment epithelial (hPRPE) cells whose genotype is MDM2 T309T. The next-generation sequencing results indicated that there was 42.51% MDM2 G309 in the edited hPRPE cells using adeno-associated viral CRISPR/Cas9. Our data showed that vitreous induced an increase in MDM2 and subsequent attenuation of p53 expression in MDM2 T309G hPRPE cells. Furthermore, our experimental results demonstrated that MDM2 T309G in hPRPE cells enhanced vitreous-induced cell proliferation and survival, suggesting that this SNP contributes to the pathogenesis of PVR. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Clustered Regularly Interspaced Short Palindromic Repeat-Dependent, Biofilm-Specific Death of Pseudomonas aeruginosa Mediated by Increased Expression of Phage-Related Genes.

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Heussler, Gary E; Cady, Kyle C; Koeppen, Katja; Bhuju, Sabin; Stanton, Bruce A; O'Toole, George A

2015-05-12

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (CRISPR/Cas) system is an adaptive immune system present in many archaea and bacteria. CRISPR/Cas systems are incredibly diverse, and there is increasing evidence of CRISPR/Cas systems playing a role in cellular functions distinct from phage immunity. Previously, our laboratory reported one such alternate function in which the type 1-F CRISPR/Cas system of the opportunistic pathogen Pseudomonas aeruginosa strain UCBPP-PA14 (abbreviated as P. aeruginosa PA14) inhibits both biofilm formation and swarming motility when the bacterium is lysogenized by the bacteriophage DMS3. In this study, we demonstrated that the presence of just the DMS3 protospacer and the protospacer-adjacent motif (PAM) on the P. aeruginosa genome is necessary and sufficient for this CRISPR-dependent loss of these group behaviors, with no requirement of additional DMS3 sequences. We also demonstrated that the interaction of the CRISPR system with the DMS3 protospacer induces expression of SOS-regulated phage-related genes, including the well-characterized pyocin operon, through the activity of the nuclease Cas3 and subsequent RecA activation. Furthermore, our data suggest that expression of the phage-related genes results in bacterial cell death on a surface due to the inability of the CRISPR-engaged strain to downregulate phage-related gene expression, while these phage-related genes have minimal impact on growth and viability under planktonic conditions. Deletion of the phage-related genes restores biofilm formation and swarming motility while still maintaining a functional CRISPR/Cas system, demonstrating that the loss of these group behaviors is an indirect effect of CRISPR self-targeting. The various CRISPR/Cas systems found in both archaea and bacteria are incredibly diverse, and advances in understanding the complex mechanisms of these varied systems has not only increased our knowledge of host

A complex of Cas proteins 5, 6, and 7 is required for the biogenesis and stability of clustered regularly interspaced short palindromic repeats (crispr)-derived rnas (crrnas) in Haloferax volcanii.

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Brendel, Jutta; Stoll, Britta; Lange, Sita J; Sharma, Kundan; Lenz, Christof; Stachler, Aris-Edda; Maier, Lisa-Katharina; Richter, Hagen; Nickel, Lisa; Schmitz, Ruth A; Randau, Lennart; Allers, Thorsten; Urlaub, Henning; Backofen, Rolf; Marchfelder, Anita

2014-03-07

The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) system is a prokaryotic defense mechanism against foreign genetic elements. A plethora of CRISPR-Cas versions exist, with more than 40 different Cas protein families and several different molecular approaches to fight the invading DNA. One of the key players in the system is the CRISPR-derived RNA (crRNA), which directs the invader-degrading Cas protein complex to the invader. The CRISPR-Cas types I and III use the Cas6 protein to generate mature crRNAs. Here, we show that the Cas6 protein is necessary for crRNA production but that additional Cas proteins that form a CRISPR-associated complex for antiviral defense (Cascade)-like complex are needed for crRNA stability in the CRISPR-Cas type I-B system in Haloferax volcanii in vivo. Deletion of the cas6 gene results in the loss of mature crRNAs and interference. However, cells that have the complete cas gene cluster (cas1-8b) removed and are transformed with the cas6 gene are not able to produce and stably maintain mature crRNAs. crRNA production and stability is rescued only if cas5, -6, and -7 are present. Mutational analysis of the cas6 gene reveals three amino acids (His-41, Gly-256, and Gly-258) that are essential for pre-crRNA cleavage, whereas the mutation of two amino acids (Ser-115 and Ser-224) leads to an increase of crRNA amounts. This is the first systematic in vivo analysis of Cas6 protein variants. In addition, we show that the H. volcanii I-B system contains a Cascade-like complex with a Cas7, Cas5, and Cas6 core that protects the crRNA.

Survey of clustered regularly interspaced short palindromic repeats and their associated Cas proteins (CRISPR/Cas) systems in multiple sequenced strains of Klebsiella pneumoniae.

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Ostria-Hernández, Martha Lorena; Sánchez-Vallejo, Carlos Javier; Ibarra, J Antonio; Castro-Escarpulli, Graciela

2015-08-04

In recent years the emergence of multidrug resistant Klebsiella pneumoniae strains has been an increasingly common event. This opportunistic species is one of the five main bacterial pathogens that cause hospital infections worldwide and multidrug resistance has been associated with the presence of high molecular weight plasmids. Plasmids are generally acquired through horizontal transfer and therefore is possible that systems that prevent the entry of foreign genetic material are inactive or absent. One of these systems is CRISPR/Cas. However, little is known regarding the clustered regularly interspaced short palindromic repeats and their associated Cas proteins (CRISPR/Cas) system in K. pneumoniae. The adaptive immune system CRISPR/Cas has been shown to limit the entry of foreign genetic elements into bacterial organisms and in some bacteria it has been shown to be involved in regulation of virulence genes. Thus in this work we used bioinformatics tools to determine the presence or absence of CRISPR/Cas systems in available K. pneumoniae genomes. The complete CRISPR/Cas system was identified in two out of the eight complete K. pneumoniae genomes sequences and in four out of the 44 available draft genomes sequences. The cas genes in these strains comprises eight cas genes similar to those found in Escherichia coli, suggesting they belong to the type I-E group, although their arrangement is slightly different. As for the CRISPR sequences, the average lengths of the direct repeats and spacers were 29 and 33 bp, respectively. BLAST searches demonstrated that 38 of the 116 spacer sequences (33%) are significantly similar to either plasmid, phage or genome sequences, while the remaining 78 sequences (67%) showed no significant similarity to other sequences. The region where the CRISPR/Cas systems were located is the same in all the Klebsiella genomes containing it, it has a syntenic architecture, and is located among genes encoding for proteins likely involved in

Correction of Hirschsprung-Associated Mutations in Human Induced Pluripotent Stem Cells Via Clustered Regularly Interspaced Short Palindromic Repeats/Cas9, Restores Neural Crest Cell Function.

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Lai, Frank Pui-Ling; Lau, Sin-Ting; Wong, John Kwong-Leong; Gui, Hongsheng; Wang, Reeson Xu; Zhou, Tingwen; Lai, Wing Hon; Tse, Hung-Fat; Tam, Paul Kwong-Hang; Garcia-Barcelo, Maria-Mercedes; Ngan, Elly Sau-Wai

2017-07-01

Hirschsprung disease is caused by failure of enteric neural crest cells (ENCCs) to fully colonize the bowel, leading to bowel obstruction and megacolon. Heterozygous mutations in the coding region of the RET gene cause a severe form of Hirschsprung disease (total colonic aganglionosis). However, 80% of HSCR patients have short-segment Hirschsprung disease (S-HSCR), which has not been associated with genetic factors. We sought to identify mutations associated with S-HSCR, and used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing system to determine how mutations affect ENCC function. We created induced pluripotent stem cell (iPSC) lines from 1 patient with total colonic aganglionosis (with the G731del mutation in RET) and from 2 patients with S-HSCR (without a RET mutation), as well as RET +/- and RET -/- iPSCs. IMR90-iPSC cells were used as the control cell line. Migration and differentiation capacities of iPSC-derived ENCCs were analyzed in differentiation and migration assays. We searched for mutation(s) associated with S-HSCR by combining genetic and transcriptome data from patient blood- and iPSC-derived ENCCs, respectively. Mutations in the iPSCs were corrected using the CRISPR/Cas9 system. ENCCs derived from all iPSC lines, but not control iPSCs, had defects in migration and neuronal lineage differentiation. RET mutations were associated with differentiation and migration defects of ENCCs in vitro. Genetic and transcriptome analyses associated a mutation in the vinculin gene (VCL M209L) with S-HSCR. CRISPR/Cas9 correction of the RET G731del and VCL M209L mutations in iPSCs restored the differentiation and migration capacities of ENCCs. We identified mutations in VCL associated with S-HSCR. Correction of this mutation in iPSC using CRISPR/Cas9 editing, as well as the RET G731del mutation that causes Hirschsprung disease with total colonic aganglionosis, restored ENCC function. Our study demonstrates how human iPSCs can

A Complex of Cas Proteins 5, 6, and 7 Is Required for the Biogenesis and Stability of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-derived RNAs (crRNAs) in Haloferax volcanii*

Science.gov (United States)

Brendel, Jutta; Stoll, Britta; Lange, Sita J.; Sharma, Kundan; Lenz, Christof; Stachler, Aris-Edda; Maier, Lisa-Katharina; Richter, Hagen; Nickel, Lisa; Schmitz, Ruth A.; Randau, Lennart; Allers, Thorsten; Urlaub, Henning; Backofen, Rolf; Marchfelder, Anita

2014-01-01

The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) system is a prokaryotic defense mechanism against foreign genetic elements. A plethora of CRISPR-Cas versions exist, with more than 40 different Cas protein families and several different molecular approaches to fight the invading DNA. One of the key players in the system is the CRISPR-derived RNA (crRNA), which directs the invader-degrading Cas protein complex to the invader. The CRISPR-Cas types I and III use the Cas6 protein to generate mature crRNAs. Here, we show that the Cas6 protein is necessary for crRNA production but that additional Cas proteins that form a CRISPR-associated complex for antiviral defense (Cascade)-like complex are needed for crRNA stability in the CRISPR-Cas type I-B system in Haloferax volcanii in vivo. Deletion of the cas6 gene results in the loss of mature crRNAs and interference. However, cells that have the complete cas gene cluster (cas1–8b) removed and are transformed with the cas6 gene are not able to produce and stably maintain mature crRNAs. crRNA production and stability is rescued only if cas5, -6, and -7 are present. Mutational analysis of the cas6 gene reveals three amino acids (His-41, Gly-256, and Gly-258) that are essential for pre-crRNA cleavage, whereas the mutation of two amino acids (Ser-115 and Ser-224) leads to an increase of crRNA amounts. This is the first systematic in vivo analysis of Cas6 protein variants. In addition, we show that the H. volcanii I-B system contains a Cascade-like complex with a Cas7, Cas5, and Cas6 core that protects the crRNA. PMID:24459147

Distribution of CRISPR spacer matches in viruses and plasmids of crenarchaeal acidothermophiles and implications for their inhibitory mechanism

DEFF Research Database (Denmark)

Shah, Shiraz Ali; Hansen, Niels R; Garrett, Roger A

2009-01-01

Transcripts from spacer sequences within chromosomal repeat clusters [CRISPRs (clusters of regularly interspaced palindromic repeats)] from archaea have been implicated in inhibiting or regulating the propagation of archaeal viruses and plasmids. For the crenarchaeal thermoacidophiles, the chromo......Transcripts from spacer sequences within chromosomal repeat clusters [CRISPRs (clusters of regularly interspaced palindromic repeats)] from archaea have been implicated in inhibiting or regulating the propagation of archaeal viruses and plasmids. For the crenarchaeal thermoacidophiles...

Clustered regulatory interspaced short palindromic repeats (CRISPR)-mediated mutagenesis and phenotype rescue by piggyBac transgenesis in a nonmodel Drosophila species.

Science.gov (United States)

Tanaka, R; Murakami, H; Ote, M; Yamamoto, D

2016-08-01

How behavioural diversity emerged in evolution is an unexplored subject in biology. To tackle this problem, genes and circuits for a behaviour need to be determined in different species for phylogenetic comparisons. The recently developed clustered regulatory interspaced short palindromic repeats/CRISPR associated protein9 (CRISPR/Cas9) system made such a challenge possible by providing the means to induce mutations in a gene of interest in any organism. Aiming at elucidating diversification in genetic and neural networks for courtship behaviour, we attempted to generate a genetic tool kit in Drosophila subobscura, a nonmodel species distantly related to the genetic model Drosophila melanogaster. Here we report the generation of yellow (y) and white mutations with the aid of the CRISPR/Cas9 system, and the rescue of the y mutant phenotype by germline transformation of the newly established y mutant fly line with a y(+) -marked piggyBac vector. This successful mutagenesis and transformation in D. subobscura open up an avenue for comprehensive genetic analyses of higher functions in this and other nonmodel Drosophila species, representing a key step toward systematic comparisons of genes and circuitries underlying behaviour amongst species. © 2016 The Royal Entomological Society.

Differential distribution of type II CRISPR-Cas systems in agricultural and nonagricultural campylobacter coli and campylobacter jejuni isolates correlates with lack of shared environments

NARCIS (Netherlands)

B.M. Pearson (Bruce M.); R.P.L. Louwen (Rogier); P. van Baarlen (Peter); A.H.M. van Vliet (Arnoud)

2015-01-01

textabstractCRISPR (clustered regularly interspaced palindromic repeats)-Cas (CRISPR-associated) systems are sequence-specific adaptive defenses against phages and plasmidswhich are widespread in prokaryotes. Herewehave studiedwhether phylogenetic relatedness or sharing of environmental niches

Differential distribution of type II CRISPR-Cas systems in agricultural and nonagricultural campylobacter coli and campylobacter jejuni isolates correlates with lack of shared environments

NARCIS (Netherlands)

Pearson, Bruce M.; Louwen, Rogier; Baarlen, Van Peter; Vliet, Van Arnoud H.M.

2015-01-01

CRISPR (clustered regularly interspaced palindromic repeats)-Cas (CRISPR-associated) systems are sequence-specific adaptive defenses against phages and plasmidswhich are widespread in prokaryotes. Herewehave studiedwhether phylogenetic relatedness or sharing of environmental niches affects the

CRISPR and the Rebirth of Synthetic Biology

NARCIS (Netherlands)

Heidari, Raheleh; Shaw, David Martin; Elger, Bernice Simone

Emergence of novel genome engineering technologies such as clustered regularly interspaced short palindromic repeat (CRISPR) has refocused attention on unresolved ethical complications of synthetic biology. Biosecurity concerns, deontological issues and human right aspects of genome editing have

A CRISPR-Cas system enhances envelope integrity mediating antibiotic resistance and inflammasome evasion

NARCIS (Netherlands)

T.R. Sampson (Timothy); B.A. Napier (Brooke); M.R. Schroeder (Max); J. Zhao (Jingshi); R.P.L. Louwen (Rogier); C.-Y. Chin (Chui-Yoke); H.K. Ratner (Hannah); A.C. Llewellyn (Anna); C.L. Jones (Crystal); H. Laroui (Hamed); D. Merlin (Didier); P. Zhou (Pei); H.P. Endtz (Hubert); D.S. Weiss (David)

2014-01-01

textabstractClustered, regularly interspaced, short palindromic repeats-CRISPR associated (CRISPR-Cas) systems defend bacteria against foreign nucleic acids, such as during bacteriophage infection and transformation, processes which cause envelope stress. It is unclear if these machineries enhance

Multiplexed CRISPR/Cas9 Genome Editing and Gene Regulation Using Csy4 in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Ferreira, Raphael; Skrekas, Christos; Nielsen, Jens

2018-01-01

Clustered regularly interspaced short palindromic repeats (CRISPR) technology has greatly accelerated the field of strain engineering. However, insufficient efforts have been made toward developing robust multiplexing tools in Saccharomyces cerevisiae. Here, we exploit the RNA processing capacity...

Design principles for nuclease-deficient CRISPR-based transcriptional regulators

DEFF Research Database (Denmark)

Jensen, Michael K.

2018-01-01

The engineering of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated proteins (Cas) continues to expand the toolkit available for genome editing, reprogramming gene regulation, genome visualization, and epigenetic studies of living organisms. In this review the ...

Unravelling the structural and mechanistic basis of CRISPR-Cas systems

NARCIS (Netherlands)

Oost, van der J.; Westra, E.R.; Jackson, R.N.; Wiedenheft, B.

2014-01-01

Bacteria and archaea have evolved sophisticated adaptive immune systems, known as CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) systems, which target and inactivate invading viruses and plasmids. Immunity is acquired by integrating short fragments

Antiviral Goes Viral : Harnessing CRISPR/Cas9 to Combat Viruses in Humans

NARCIS (Netherlands)

Soppe, Jasper Adriaan; Lebbink, Robert Jan

2017-01-01

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems are RNA-guided sequence-specific prokaryotic antiviral immune systems. In prokaryotes, small RNA molecules guide Cas effector endonucleases to invading foreign genetic elements in a

Structural basis for CRISPR RNA-guided DNA recognition by Cascade

NARCIS (Netherlands)

Jore, M.M.; Lundgren, N.M.J.; Duijn, van E.; Bultema, J.B.; Westra, E.R.; Oost, van der J.; Brouns, S.J.J.; Beijer, M.R.

2011-01-01

The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and

Structural basis for CRISPR RNA-guided DNA recognition by Cascade

NARCIS (Netherlands)

Jore, Matthijs M.; Lundgren, Magnus; van Duijn, Esther; Bultema, Jelle B.; Westra, Edze R.; Waghmare, Sakharam P.; Wiedenheft, Blake; Pul, Uemit; Wurm, Reinhild; Wagner, Rolf; Beijer, Marieke R.; Barendregt, Arjan; Zhou, Kaihong; Snijders, Ambrosius P. L.; Dickman, Mark J.; Doudna, Jennifer A.; Boekema, Egbert J.; Heck, Albert J. R.; van der Oost, John; Brouns, Stan J. J.; Pul, Ümit

The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and

Type I-E CRISPR-cas systems discriminate target from non-target DNA through base pairing-independent PAM recognition

NARCIS (Netherlands)

Westra, E.R.; Semenova, E.; Datsenko, K.A.; Jackson, R.N.; Wiedenheft, B.; Severinov, K.; Brouns, S.J.J.

2013-01-01

Discriminating self and non-self is a universal requirement of immune systems. Adaptive immune systems in prokaryotes are centered around repetitive loci called CRISPRs (clustered regularly interspaced short palindromic repeat), into which invader DNA fragments are incorporated. CRISPR transcripts

Targeted Genome Regulation and Editing in Plants

KAUST Repository

Piatek, Agnieszka Anna

2016-01-01

for bioengineering applications. However, the TALE system is limited by the requirement to re-engineer one or two proteins for each new target sequence. Recently, the clustered regularly interspaced palindromic repeats (CRISPR)/ CRISPR associated 9 (Cas9) has been

In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration

KAUST Repository

Suzuki, Keiichiro; Tsunekawa, Yuji; Herná ndez-Bení tez, Reyna; Wu, Jun; Zhu, Jie; Kim, Euiseok J.; Hatanaka, Fumiyuki; Yamamoto, Mako; Araoka, Toshikazu; Li, Zhe; Kurita, Masakazu; Hishida, Tomoaki; Li, Mo; Aizawa, Emi; Guo, Shicheng; Chen, Song; Goebl, April; Soligalla, Rupa Devi; Qu, Jing; Jiang, Tingshuai; Fu, Xin; Jafari, Maryam; Esteban, Concepcion Rodriguez; Berggren, W. Travis; Lajara, Jeronimo; Nuñ ez-Delicado, Estrella; Guillen, Pedro; Campistol, Josep M.; Matsuzaki, Fumio; Liu, Guang-Hui; Magistretti, Pierre J.; Zhang, Kun; Callaway, Edward M.; Zhang, Kang; Belmonte, Juan Carlos Izpisua

2016-01-01

regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9)3, 4 technology, here we devise a homology-independent targeted integration (HITI) strategy, which allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and, more

Crystal structure of the CRISPR RNA–guided surveillance complex from Escherichia coli

NARCIS (Netherlands)

Jackson, R.N.; Golden, S.M.; Erp, P.B.; Carter, J.; Westra, E.R.; Brouns, S.J.J.; Oost, van der J.; Terwilliger, T.C.; Read, R.J.; Wiedenheft, B.

2014-01-01

Clustered regularly interspaced short palindromic repeats (CRISPRs) are essential components of RNA-guided adaptive immune systems that protect bacteria and archaea from viruses and plasmids. In Escherichia coli, short CRISPR-derived RNAs (crRNAs) assemble into a 405-kilodalton multisubunit

Degenerate target sites mediate rapid primed CRISPR adaptation

NARCIS (Netherlands)

Fineran, P.C.; Gerritzen, M.J.; Suarez-Diez, M.; Kunne, T.; Boekhorst, J.; Hijum, S.A.F.T. van; Staals, R.H.G.; Brouns, S.J.

2014-01-01

Prokaryotes encode adaptive immune systems, called CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated), to provide resistance against mobile invaders, such as viruses and plasmids. Host immunity is based on incorporation of invader DNA sequences in a memory locus

Degenerate target sites mediate rapid primed CRISPR adaptation

NARCIS (Netherlands)

Fineran, P.C.; Gerritzen, M.J.H.; Suarez Diez, M.; Künne, T.A.; Boekhorst, J.; Hijum, van S.A.F.T.; Staals, R.H.J.; Brouns, S.J.J.

2014-01-01

Prokaryotes encode adaptive immune systems, called CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR associated), to provide resistance against mobile invaders, such as viruses and plasmids. Host immunity is based on incorporation of invader DNA sequences in a memory locus

CRISPR and piRNAs: Fundamental Mechanisms and Key Applications of the Next Generation of Molecular Technologies in the Field of Toxicology

Science.gov (United States)

" Exploration into the roles of genes, the proteins that they encode, and the functions that they carry out within the cell is a founding pillar in the field of toxicology. Recent breakthroughs in clustered, regularly interspaced, short palindromic repeat (CRISPR) technology...

Temperature-dependent sex-reversal by a transformer-2 gene-edited mutation in the spotted wing drosophila, Drosophila suzukii

Science.gov (United States)

Female to male sex reversal was achieved in an emerging agricultural insect pest, Drosophila suzukii, by creating a temperature-sensitive point mutation in the sex-determination gene, transformer-2 (tra-2) using CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/ CRISPR-associated) hom...

Structural biology. Structures of the CRISPR-Cmr complex reveal mode of RNA target positioning

NARCIS (Netherlands)

Taylor, D.W.; Zhu, Y.; Staals, R.H.J.; Kornfeld, J.E.; Shinkai, A.; Oost, van der J.; Nogales, E.; Doudna, J.A.

2015-01-01

Adaptive immunity in bacteria involves RNA-guided surveillance complexes that use CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR RNAs (crRNAs) to target invasive nucleic acids for degradation. Whereas type I and type II CRISPR-Cas

Exploiting off-targeting in guide-RNAs for CRISPR systems for simultaneous editing of multiple genes

DEFF Research Database (Denmark)

Ferreira, Raphael; Gatto, Francesco; Nielsen, Jens

2017-01-01

Bioinformatics tools to design guide-RNAs (gRNAs) in Clustered Regularly Interspaced Short Palindromic Repeats systems mostly focused on minimizing off-targeting to enhance efficacy of genome editing. However, there are circumstances in which off-targeting might be desirable to target multiple ge...

Evolution and classification of the CRISPR-Cas systems

NARCIS (Netherlands)

Makarova, K.S.; Brouns, S.J.J.; Oost, van der J.

2011-01-01

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) modules are adaptive immunity systems that are present in many archaea and bacteria. These defence systems are encoded by operons that have an extraordinarily diverse architecture and a high rate of

CRISPR-Cas9 Can Inhibit HIV-1 Replication but NHEJ Repair Facilitates Virus Escape

NARCIS (Netherlands)

Wang, Gang; Zhao, Na; Berkhout, Ben; Das, Atze T.

2016-01-01

Several recent studies demonstrated that the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 can be used for guide RNA (gRNA)-directed, sequence-specific cleavage of HIV proviral DNA in infected cells. We here demonstrate profound inhibition of HIV-1

Small CRISPR RNAs guide antiviral defense in prokaryotes

NARCIS (Netherlands)

Brouns, S.J.J.; Jore, M.M.; Lundgren, M.; Westra, E.R.; Slijkhuis, R.J.; Snijders, A.P.; Dickman, M.J.; Makarova, K.S.; Koonin, E.V.; Oost, van der J.

2008-01-01

Prokaryotes acquire virus resistance by integrating short fragments of viral nucleic acid into clusters of regularly interspaced short palindromic repeats (CRISPRs). Here we show how virus-derived sequences contained in CRISPRs are used by CRISPR-associated (Cas) proteins from the host to mediate an

The Role of CRISPR-Cas Systems in Virulence of Pathogenic Bacteria

NARCIS (Netherlands)

Louwen, R.; Staals, R.H.J.; Endtz, H.P.; Baarlen, van P.; Oost, van der J.

2014-01-01

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are present in many bacterial and archaeal genomes. Since the discovery of the typical CRISPR loci in the 1980s, well before their physiological role was revealed, their variable sequences have been

Cas4 Facilitates PAM-Compatible Spacer Selection during CRISPR Adaptation

NARCIS (Netherlands)

Kieper, Sebastian N.; Almendros, Cristóbal; Behler, Juliane; McKenzie, Rebecca E.; Nobrega, Franklin L.; Haagsma, Anna C.; Vink, Jochem N.A.; Hess, Wolfgang R.; Brouns, Stan J.J.

2018-01-01

CRISPR-Cas systems adapt their immunological memory against their invaders by integrating short DNA fragments into clustered regularly interspaced short palindromic repeat (CRISPR) loci. While Cas1 and Cas2 make up the core machinery of the CRISPR integration process, various class I and II

Postnatal Cardiac Gene Editing Using CRISPR/Cas9 With AAV9-Mediated Delivery of Short Guide RNAs Results in Mosaic Gene Disruption

NARCIS (Netherlands)

Johansen, Anne Katrine; Molenaar, Bas; Versteeg, Danielle; Leitoguinho, Ana Rita; Demkes, Charlotte; Spanjaard, Bastiaan; de Ruiter, Hesther; Akbari Moqadam, Farhad; Kooijman, Lieneke; Zentilin, Lorena; Giacca, Mauro; van Rooij, Eva

2017-01-01

RATIONALE: CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9)-based DNA editing has rapidly evolved as an attractive tool to modify the genome. Although CRISPR/Cas9 has been extensively used to manipulate the germline in zygotes, its application in

CRISPR-Cas9 gene editing

NARCIS (Netherlands)

Oude Blenke, Erik; Evers, Martijn J.W.; Mastrobattista, Enrico; Oost, van der John

2016-01-01

The CRISPR-Cas9 gene editing system has taken the biomedical science field by storm, initiating rumors about future Nobel Prizes and heating up a fierce patent war, but also making significant scientific impact. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), together with

Analyses of the Sequence and Structural Properties Corresponding to Pentapeptide and Large Palindromes in Proteins.

Directory of Open Access Journals (Sweden)

Settu Sridhar

Full Text Available The analyses of 3967 representative proteins selected from the Protein Data Bank revealed the presence of 2803 pentapeptide and large palindrome sequences with known secondary structure conformation. These represent 2014 unique palindrome sequences. 60% palindromes are not associated with any regular secondary structure and 28% are in helix conformation, 11% in strand conformation and 1% in the coil conformation. The average solvent accessibility values are in the range between 0-155.28 Å2 suggesting that the palindromes in proteins can be either buried, exposed to the solvent or share an intermittent property. The number of residue neighborhood contacts defined by interactions ≤ 3.2 Ǻ is in the range between 0-29 residues. Palindromes of the same length in helix, strand and coil conformation are associated with different amino acid residue preferences at the individual positions. Nearly, 20% palindromes interact with catalytic/active site residues, ligand or metal ions in proteins and may therefore be important for function in the corresponding protein. The average hydrophobicity values for the pentapeptide and large palindromes range between -4.3 to +4.32 and the number of palindromes is almost equally distributed between the negative and positive hydrophobicity values. The palindromes represent 107 different protein families and the hydrolases, transferases, oxidoreductases and lyases contain relatively large number of palindromes.

Cas4 Facilitates PAM-Compatible Spacer Selection during CRISPR Adaptation

NARCIS (Netherlands)

Kieper, S.N.; Almendros, Cristóbal; Behler, Juliane; McKenzie, R.E.; Luzia De Nóbrega, F.; van Eijkeren-Haagsma, A.C.; Vink, J.N.A.; Hess, Wolfgang R.; Brouns, S.J.J.

2018-01-01

CRISPR-Cas systems adapt their immunological memory against their invaders by integrating short DNA fragments into clustered regularly interspaced short palindromic repeat (CRISPR) loci. While Cas1 and Cas2 make up the core machinery of the CRISPR integration process, various class I and II

Seamless gene editing in Aspergillus species, using CRISPR-Cas9

DEFF Research Database (Denmark)

Nødvig, Christina Spuur; Hoof, Jakob Blæsbjerg; Mortensen, Uffe Hasbro

there are several obstacles slowing down the process. The harnessing of the prokaryotic and archaeal immune mechanism CRISPR (clustered regularly interspaced short palindromic repeats) as a tool for genetic engineering in eukaryotes, has proved to be a powerful technology. CRISPR/Cas9 introduces specific DNA double...

CRISPR technologies for bacterial systems: Current achievements and future directions

DEFF Research Database (Denmark)

Choi, Kyeong Rok; Lee, Sang Yup

2016-01-01

Throughout the decades of its history, the advances in bacteria-based bio-industries have coincided with great leaps in strain engineering technologies. Recently unveiled clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) systems are now revolution......Throughout the decades of its history, the advances in bacteria-based bio-industries have coincided with great leaps in strain engineering technologies. Recently unveiled clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) systems are now...... revolutionizing biotechnology as well as biology. Diverse technologies have been derived from CRISPR/Cas systems in bacteria, yet the applications unfortunately have not been actively employed in bacteria as extensively as in eukaryotic organisms. A recent trend of engineering less explored strains in industrial...... microbiology-metabolic engineering, synthetic biology, and other related disciplines-is demanding facile yet robust tools, and various CRISPR technologies have potential to cater to the demands. Here, we briefly review the science in CRISPR/Cas systems and the milestone inventions that enabled numerous CRISPR...

A Golden Gate-based Protocol for Assembly of Multiplexed gRNA Expression Arrays for CRISPR/Cas9

DEFF Research Database (Denmark)

Vad-Nielsen, Johan; Lin, Lin; Jensen, Kristopher Torp

2016-01-01

The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) has become the most broadly used and powerful tool for genome editing. Many applications of CRISPR-Cas9 require the delivery of multiple small guide RNAs (gRNAs) into the same cell in order to achieve...

Application of CRISPR/Cas9 Genome Editing to Improve Recombinant Protein Production in CHO Cells

DEFF Research Database (Denmark)

Grav, Lise Marie; Julie la Cour Karottki, Karen; Lee, Jae Seong

2017-01-01

and yields. In this chapter, we present our protocol on how to use the genome editing tool Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) to knockout engineering target genes in CHO cells. As an example, we refer to the glutamine synthetase (GS...

Discovery of "Escherichia coli" CRISPR Sequences in an Undergraduate Laboratory

Science.gov (United States)

Militello, Kevin T.; Lazatin, Justine C.

2017-01-01

Clustered regularly interspaced short palindromic repeats (CRISPRs) represent a novel type of adaptive immune system found in eubacteria and archaebacteria. CRISPRs have recently generated a lot of attention due to their unique ability to catalog foreign nucleic acids, their ability to destroy foreign nucleic acids in a mechanism that shares some…

The on–off switch of CRISPR immunity against phages in Escherichia coli

OpenAIRE

Mojica, Francisco J.M.; Díez-Villaseñor, César

2010-01-01

CRISPR/CAS is a novel prokaryotic defence system against cell invaders. A typical CRISPR/CAS system consists of one or more clusters of regularly interspaced short, often palindromic, repeats (CRISPR) followed at one side by an adenine- and thymine-rich (AT-rich) leader sequence, and a variable set of CAS (CRISPR associated) genes.

CRISPR/Cas system for yeast genome engineering: advances and applications

DEFF Research Database (Denmark)

Stovicek, Vratislav; Holkenbrink, Carina; Borodina, Irina

2017-01-01

The methods based on the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system have quickly gained popularity for genome editing and transcriptional regulation in many organisms, including yeast. This review aims to provide a comprehensive overview...... of CRISPR application for different yeast species: from basic principles and genetic design to applications....

CRISPR-Cpf1: A New Tool for Plant Genome Editing

KAUST Repository

Zaidi, Syed Shan-e-Ali; Mahfouz, Magdy M.; Mansoor, Shahid

2017-01-01

Clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated proteins (CRISPR-Cas), a groundbreaking genome-engineering tool, has facilitated targeted trait improvement in plants. Recently, CRISPR-CRISPR from Prevotella and Francisella 1 (Cpf1) has emerged as a new tool for efficient genome editing, including DNA-free editing in plants, with higher efficiency, specificity, and potentially wider applications than CRISPR-Cas9.

CRISPR-Cpf1: A New Tool for Plant Genome Editing

KAUST Repository

Zaidi, Syed Shan-e-Ali

2017-05-19

Clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated proteins (CRISPR-Cas), a groundbreaking genome-engineering tool, has facilitated targeted trait improvement in plants. Recently, CRISPR-CRISPR from Prevotella and Francisella 1 (Cpf1) has emerged as a new tool for efficient genome editing, including DNA-free editing in plants, with higher efficiency, specificity, and potentially wider applications than CRISPR-Cas9.

System-level perturbations of cell metabolism using CRISPR/Cas9

DEFF Research Database (Denmark)

Jakociunas, Tadas; Jensen, Michael Krogh; Keasling, Jay

2017-01-01

CRISPR/Cas9 (clustered regularly interspaced palindromic repeats and the associated protein Cas9) techniques have made genome engineering and transcriptional reprogramming studies more advanced and cost-effective. For metabolic engineering purposes, the CRISPR-based tools have been applied...... previously possible. In this mini-review we highlight recent studies adopting CRISPR/Cas9 for systems-level perturbations and model-guided metabolic engineering....

CRISPR-Cas and Restriction-Modification Act Additively against Conjugative Antibiotic Resistance Plasmid Transfer in Enterococcus faecalis

OpenAIRE

Price, Valerie J.; Huo, Wenwen; Sharifi, Ardalan; Palmer, Kelli L.

2016-01-01

ABSTRACT Enterococcus faecalis is an opportunistic pathogen and a leading cause of nosocomial infections. Conjugative pheromone-responsive plasmids are narrow-host-range mobile genetic elements (MGEs) that are rapid disseminators of antibiotic resistance in the faecalis species. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas and restriction-modification confer acquired and innate immunity, respectively, against MGE acquisition in bacteria. Most multidrug-resistant E.?fa...

Desktop Genetics

OpenAIRE

Hough, Soren H; Ajetunmobi, Ayokunmi; Brody, Leigh; Humphryes-Kirilov, Neil; Perello, Edward

2016-01-01

Desktop Genetics is a bioinformatics company building a gene-editing platform for personalized medicine. The company works with scientists around the world to design and execute state-of-the-art clustered regularly interspaced short palindromic repeats (CRISPR) experiments. Desktop Genetics feeds the lessons learned about experimental intent, single-guide RNA design and data from international genomics projects into a novel CRISPR artificial intelligence system. We believe that machine learni...

Draft Genome Sequence of Ezakiella peruensis Strain M6.X2, a Human Gut Gram-Positive Anaerobic Coccus.

Science.gov (United States)

Diop, Awa; Diop, Khoudia; Tomei, Enora; Raoult, Didier; Fenollar, Florence; Fournier, Pierre-Edouard

2018-03-01

We report here the draft genome sequence of Ezakiella peruensis strain M6.X2 T The draft genome is 1,672,788 bp long and harbors 1,589 predicted protein-encoding genes, including 26 antibiotic resistance genes with 1 gene encoding vancomycin resistance. The genome also exhibits 1 clustered regularly interspaced short palindromic repeat region and 333 genes acquired by horizontal gene transfer. Copyright © 2018 Diop et al.

Recent advances in functional perturbation and genome editing techniques in studying sea urchin development.

Science.gov (United States)

Cui, Miao; Lin, Che-Yi; Su, Yi-Hsien

2017-09-01

Studies on the gene regulatory networks (GRNs) of sea urchin embryos have provided a basic understanding of the molecular mechanisms controlling animal development. The causal links in GRNs have been verified experimentally through perturbation of gene functions. Microinjection of antisense morpholino oligonucleotides (MOs) into the egg is the most widely used approach for gene knockdown in sea urchin embryos. The modification of MOs into a membrane-permeable form (vivo-MOs) has allowed gene knockdown at later developmental stages. Recent advances in genome editing tools, such as zinc-finger nucleases, transcription activator-like effector-based nucleases and the clustered regularly interspaced short palindromic repeat/clustered regularly interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9) system, have provided methods for gene knockout in sea urchins. Here, we review the use of vivo-MOs and genome editing tools in sea urchin studies since the publication of its genome in 2006. Various applications of the CRISPR/Cas9 system and their potential in studying sea urchin development are also discussed. These new tools will provide more sophisticated experimental methods for studying sea urchin development. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Cas9-triggered chain ablation of cas9 as a gene drive brake

OpenAIRE

Wu, Bing; Luo, Liqun; Gao, Xiaojing J.

2016-01-01

With the advent of clustered, regularly interspaced, short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) technology, researchers can construct gene drives that can bias the inheritance of edited alleles to alter entire populations. As demonstrated with the mutagenic chain reaction in Drosophila4, the CRISPR-Cas9 system can propagate genomic modification together with the genome-editing machinery itself. Although gene drives might have the potential to control insect-borne di...

Presence of Type I-F CRISPR/Cas systems is associated with antimicrobial susceptibility in Escherichia coli.

OpenAIRE

Aydin, Seyid; Personne, Yoann; Newire, Enas; Laverick, Rebecca; Russell, Oliver; Roberts, Adam; Enne, Virve I

2017-01-01

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated cas genes are sequence-specific DNA nuclease systems found in bacteria and archaea. CRISPR/Cas systems use RNA transcripts of previously acquired DNA (spacers) to target invading genetic elements with the same sequence, including plasmids. In this research we studied the relationship between CRISPR/Cas systems and multidrug resistance in Escherichia coli . The presence of Type I-E and Type I-F CRISPR syste...

Genetic engineering of a temperate phage-based delivery system for CRISPR/Cas9 antimicrobials against Staphylococcus aureus

OpenAIRE

Park, Joo Youn; Moon, Bo Youn; Park, Juw Won; Thornton, Justin A.; Park, Yong Ho; Seo, Keun Seok

2017-01-01

Discovery of clustered, regularly interspaced, short palindromic repeats and the Cas9 RNA-guided nuclease (CRISPR/Cas9) system provides a new opportunity to create programmable gene-specific antimicrobials that are far less likely to drive resistance than conventional antibiotics. However, the practical therapeutic use of CRISPR/Cas9 is still questionable due to current shortcomings in phage-based delivery systems such as inefficient delivery, narrow host range, and potential transfer of viru...

?Off-Spotter?: very fast and exhaustive enumeration of genomic lookalikes for designing CRISPR/Cas guide RNAs

OpenAIRE

Pliatsika, Venetia; Rigoutsos, Isidore

2015-01-01

Background CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated nucleases) is a powerful component of the prokaryotic immune system that has been adapted for targeted genetic engineering in higher organisms. A key element of CRISPR/Cas is the ?guide? RNA (gRNA) that is ~20 nucleotides (nts) in length and designed to be complementary to the intended target site. An integral requirement of the CRISPR/Cas system is that the target site be followed by a protospa...

Researchers use Modified CRISPR Systems to Modulate Gene Expression on a Genomic Scale | Office of Cancer Genomics

Science.gov (United States)

The genetic engineering system, clustered regularly interspaced short palindromic repeats (CRISPR), has conventionally been used to inactivate genes by making targeted double stranded cuts in DNA. While CRISPR is a useful tool, it can only be used to create loss-of-function modifications and often causes off-target effects due to the disruptive mechanism by which it works. CTD2 researchers at the University of California, San Francisco recently addressed these shortcomings in a publication in Cell.

Chromosomal Targeting by the Type III-A CRISPR-Cas System Can Reshape Genomes in Staphylococcus aureus

OpenAIRE

Guan, Jing; Wang, Wanying; Sun, Baolin

2017-01-01

ABSTRACT CRISPR-Cas (clustered regularly interspaced short palindromic repeat [CRISPR]-CRISPR-associated protein [Cas]) systems can provide protection against invading genetic elements by using CRISPR RNAs (crRNAs) as a guide to locate and degrade the target DNA. CRISPR-Cas systems have been classified into two classes and five types according to the content of cas genes. Previous studies have indicated that CRISPR-Cas systems can avoid viral infection and block plasmid transfer. Here we show...

The genome editing revolution: A CRISPR-Cas TALE off-target story.

Science.gov (United States)

Stella, Stefano; Montoya, Guillermo

2016-07-01

In the last 10 years, we have witnessed a blooming of targeted genome editing systems and applications. The area was revolutionized by the discovery and characterization of the transcription activator-like effector proteins, which are easier to engineer to target new DNA sequences than the previously available DNA binding templates, zinc fingers and meganucleases. Recently, the area experimented a quantum leap because of the introduction of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system (clustered regularly interspaced short palindromic sequence). This ribonucleoprotein complex protects bacteria from invading DNAs, and it was adapted to be used in genome editing. The CRISPR ribonucleic acid (RNA) molecule guides to the specific DNA site the Cas9 nuclease to cleave the DNA target. Two years and more than 1000 publications later, the CRISPR-Cas system has become the main tool for genome editing in many laboratories. Currently the targeted genome editing technology has been used in many fields and may be a possible approach for human gene therapy. Furthermore, it can also be used to modifying the genomes of model organisms for studying human pathways or to improve key organisms for biotechnological applications, such as plants, livestock genome as well as yeasts and bacterial strains. © 2016 The Authors. BioEssays published by WILEY Periodicals, Inc.

Desktop Genetics.

Science.gov (United States)

Hough, Soren H; Ajetunmobi, Ayokunmi; Brody, Leigh; Humphryes-Kirilov, Neil; Perello, Edward

2016-11-01

Desktop Genetics is a bioinformatics company building a gene-editing platform for personalized medicine. The company works with scientists around the world to design and execute state-of-the-art clustered regularly interspaced short palindromic repeats (CRISPR) experiments. Desktop Genetics feeds the lessons learned about experimental intent, single-guide RNA design and data from international genomics projects into a novel CRISPR artificial intelligence system. We believe that machine learning techniques can transform this information into a cognitive therapeutic development tool that will revolutionize medicine.

Cas4 Facilitates PAM-Compatible Spacer Selection during CRISPR Adaptation

OpenAIRE

Sebastian N. Kieper; Cristóbal Almendros; Juliane Behler; Rebecca E. McKenzie; Franklin L. Nobrega; Anna C. Haagsma; Jochem N.A. Vink; Wolfgang R. Hess; Stan J.J. Brouns

2018-01-01

Summary: CRISPR-Cas systems adapt their immunological memory against their invaders by integrating short DNA fragments into clustered regularly interspaced short palindromic repeat (CRISPR) loci. While Cas1 and Cas2 make up the core machinery of the CRISPR integration process, various class I and II CRISPR-Cas systems encode Cas4 proteins for which the role is unknown. Here, we introduced the CRISPR adaptation genes cas1, cas2, and cas4 from the type I-D CRISPR-Cas system of Synechocystis sp....

CRISPR/Cas9: at the cutting edge of hepatology

Science.gov (United States)

Pankowicz, Francis P; Jarrett, Kelsey E; Lagor, William R; Bissig, Karl-Dimiter

2018-01-01

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome engineering has revolutionised biomedical science and we are standing on the cusp of medical transformation. The therapeutic potential of this technology is tremendous, however, its translation to the clinic will be challenging. In this article, we review recent progress using this genome editing technology and explore its potential uses in studying and treating diseases of the liver. We discuss the development of new research tools and animal models as well as potential clinical applications, strategies and challenges. PMID:28487442

Use of CRISPR/Cas Genome Editing Technology for Targeted Mutagenesis in Rice.

Science.gov (United States)

Xu, Rongfang; Wei, Pengcheng; Yang, Jianbo

2017-01-01

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) system is a newly emerging mutagenesis (gene-editing) tool in genetic engineering. Among the agriculturally important crops, several genes have been successfully mutated by the system, and some agronomic important traits have been rapidly generated, which indicates the potential applications in both scientific research and plant breeding. In this chapter, we describe a standard gene-editing procedure to effectively target rice genes and to make specific rice mutants using the CRISPR/Cas9 system mediated by Agrobacterium transformation.

Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Triggered Isothermal Amplification for Site-Specific Nucleic Acid Detection.

Science.gov (United States)

Huang, Mengqi; Zhou, Xiaoming; Wang, Huiying; Xing, Da

2018-02-06

A novel CRISPR/Cas9 triggered isothermal exponential amplification reaction (CAS-EXPAR) strategy based on CRISPR/Cas9 cleavage and nicking endonuclease (NEase) mediated nucleic acids amplification was developed for rapid and site-specific nucleic acid detection. CAS-EXPAR was primed by the target DNA fragment produced by cleavage of CRISPR/Cas9, and the amplification reaction performed cyclically to generate a large number of DNA replicates which were detected using a real-time fluorescence monitoring method. This strategy that combines the advantages of CRISPR/Cas9 and exponential amplification showed high specificity as well as rapid amplification kinetics. Unlike conventional nucleic acids amplification reactions, CAS-EXPAR does not require exogenous primers, which often cause target-independent amplification. Instead, primers are first generated by Cas9/sgRNA directed site-specific cleavage of target and accumulated during the reaction. It was demonstrated this strategy gave a detection limit of 0.82 amol and showed excellent specificity in discriminating single-base mismatch. Moreover, the applicability of this method to detect DNA methylation and L. monocytogenes total RNA was also verified. Therefore, CAS-EXPAR may provide a new paradigm for efficient nucleic acid amplification and hold the potential for molecular diagnostic applications.

Characteristics of palindromic sequences in DNA of the sea urchin Stronglyocentrotus intermedius

International Nuclear Information System (INIS)

Brykov, V.A.; Kukhlevskii, A.D.

1986-01-01

The fraction of palindromic sequences in the nuclear DNA of the sea urchin S. intermedius was characterized. Using chromatography on hydroxyapatite and treatment with S1 nuclease, it was shown that the fraction of palindromic sequences more than doubles when the sodium concentration in solution is increased or the temperature of reassociation is lowered. The increase is due to the involvement of inverted repeats in reassociation, which are characterized by a substantial nonhomologous character and/or the presence of an extended intervening DNA sequence. It was found by the method of reassociation of a nicked palindrome fraction with an excess of total homologous DNA that most of the inverted repeats in the sea urchin genome are unique sequences. The complexity of the palindrome fraction was estimated at 8.2 x 10 7 nucleotide pairs, and the number of palindromes per haploid genome ∼ 500,000

Production of genome-edited pluripotent stem cells and mice by CRISPR/Cas.

Science.gov (United States)

Horii, Takuro; Hatada, Izuho

2016-01-01

Clustered regularly at interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nucleases, so-called CRISPR/Cas, was recently developed as an epoch-making genome engineering technology. This system only requires Cas9 nuclease and single-guide RNA complementary to a target locus. CRISPR/Cas enables the generation of knockout cells and animals in a single step. This system can also be used to generate multiple mutations and knockin in a single step, which is not possible using other methods. In this review, we provide an overview of genome editing by CRISPR/Cas in pluripotent stem cells and mice.

System-level perturbations of cell metabolism using CRISPR/Cas9

Energy Technology Data Exchange (ETDEWEB)

Jakočiūnas, Tadas [Technical Univ. of Denmark, Lyngby (Denmark); Jensen, Michael K. [Technical Univ. of Denmark, Lyngby (Denmark); Keasling, Jay D. [Technical Univ. of Denmark, Lyngby (Denmark); Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)

2017-03-30

CRISPR/Cas9 (clustered regularly interspaced palindromic repeats and the associated protein Cas9) techniques have made genome engineering and transcriptional reprogramming studies much more advanced and cost-effective. For metabolic engineering purposes, the CRISPR-based tools have been applied to single and multiplex pathway modifications and transcriptional regulations. The effectiveness of these tools allows researchers to implement genome-wide perturbations, test model-guided genome editing strategies, and perform transcriptional reprogramming perturbations in a more advanced manner than previously possible. In this mini-review we highlight recent studies adopting CRISPR/Cas9 for systems-level perturbations and model-guided metabolic engineering.

CRISPR/Cas9 for Human Genome Engineering and Disease Research.

Science.gov (United States)

Xiong, Xin; Chen, Meng; Lim, Wendell A; Zhao, Dehua; Qi, Lei S

2016-08-31

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system, a versatile RNA-guided DNA targeting platform, has been revolutionizing our ability to modify, manipulate, and visualize the human genome, which greatly advances both biological research and therapeutics development. Here, we review the current development of CRISPR/Cas9 technologies for gene editing, transcription regulation, genome imaging, and epigenetic modification. We discuss the broad application of this system to the study of functional genomics, especially genome-wide genetic screening, and to therapeutics development, including establishing disease models, correcting defective genetic mutations, and treating diseases.

Archaeal CRISPR-based immune systems

DEFF Research Database (Denmark)

Garrett, Roger A; Vestergaard, Gisle Alberg; Shah, Shiraz Ali

2011-01-01

CRISPR (clustered regularly interspaced short palindromic repeats)-based immune systems are essentially modular with three primary functions: the excision and integration of new spacers, the processing of CRISPR transcripts to yield mature CRISPR RNAs (crRNAs), and the targeting and cleavage...... of foreign nucleic acid. The primary target appears to be the DNA of foreign genetic elements, but the CRISPR/Cmr system that is widespread amongst archaea also specifically targets and cleaves RNA in vitro. The archaeal CRISPR systems tend to be both diverse and complex. Here we examine evidence...... of CRISPR loci and the evidence for intergenomic exchange of CRISPR systems....

[Overview of patents on targeted genome editing technologies and their implications for innovation and entrepreneurship education in universities].

Science.gov (United States)

Fan, Xiang-yu; Lin, Yan-ping; Liao, Guo-jian; Xie, Jian-ping

2015-12-01

Zinc finger nuclease, transcription activator-like effector nuclease, and clustered regularly interspaced short palindromic repeats/Cas9 nuclease are important targeted genome editing technologies. They have great significance in scientific research and applications on aspects of functional genomics research, species improvement, disease prevention and gene therapy. There are past or ongoing disputes over ownership of the intellectual property behind every technology. In this review, we summarize the patents on these three targeted genome editing technologies in order to provide some reference for developing genome editing technologies with self-owned intellectual property rights and some implications for current innovation and entrepreneurship education in universities.

The JET vacuum interspace system

International Nuclear Information System (INIS)

Orchard, J.; Scales, S.

1999-01-01

In the past JET has suffered from a number of vacuum leaks on components such as bellows, windows and feedthroughs due, in part, to the adverse conditions, including high mechanical forces, which may prevail during plasma operation. Therefore before the recent Tritium experiments on JET it was deemed prudent to manufacture and install items with a secondary containment or interspace in order to minimise the effect of failure of the primary vacuum barrier on both the leak integrity of the machine and the outcome of the experiments. This paper describes the philosophy, logistics, method and implementation of an integrated connection and monitoring system on the 330 interspaces currently in position on the JET machine. Using the JET leak database comparisons are drawn of leak failure rates of the components allied to the number of operational hours, prior to the system being present and after installation and commissioning, and the case of detection compared to the previous situation. An argument is also presented on the feasibility and adaptability of this system to any large complex machine and the benefits to be obtained in reduction of leaks and operational down time. (author)

Interspace modification of titania-nanorod arrays for efficient mesoscopic perovskite solar cells

International Nuclear Information System (INIS)

Chen, Peng; Jin, Zhixin; Wang, Yinglin; Wang, Meiqi; Chen, Shixin; Zhang, Yang; Wang, Lingling; Zhang, Xintong; Liu, Yichun

2017-01-01

Highlights: • The fabrication of perovskite solar cells utilizing TiO_2 NR arrays. • Investigation of the interspace effect of TiO_2 NR on perovskite layer. • Understanding of the balance between perovskite capping layer and pore filling. - Abstract: Morphology of electron transport layers (ETLs) has an important influence on the device architecture and electronic processes of mesostructured solar cells. In this work, we thoroughly investigated the effect of the interspace of TiO_2 nanorod (NR) arrays on the photovoltaic performance of the perovskite solar cells (PSCs). Along with the interspace in TiO_2-NR arrays increasing, the thickness as well as the crystal size of perovskite capping layer are reduced accordingly, and the filling of perovskite in the channel becomes incomplete. Electrochemical impedance spectroscopy measurements reveal that this variation of perovskite absorber layer, induced by interspace of TiO_2 NR arrays, causes the change of charge recombination process at the TiO_2/perovskite interface, suggesting that a balance between capping layer and the perovskite filling is critical to obtain high charge collection efficiency of PSCs. A power conversion efficiency of 10.3% could be achieved through careful optimization of interspace in TiO_2-NR arrays. Our research will shed light on the morphology control of ETLs with 1D structure for heterojunction solar cells fabricated by solution-deposited method.

Schroedinger operators with Rudin-Shapiro potentials are not palindromic

International Nuclear Information System (INIS)

Allouche, J.

1997-01-01

We prove a conjecture of A. Hof, O. Knill and B. Simon [Commun. Math. Phys. 174, 149 endash 159 (1995)] by showing that the Rudin-Shapiro sequence is not palindromic, i.e., does not contain arbitrarily long palindromes. We prove actually this property for all paperfolding sequences and all Rudin-Shapiro sequences deduced from paperfolding sequences. As a consequence and as guessed by the above authors, their method cannot be used for establishing that discrete Schroedinger operators with Rudin-Shapiro potentials have a purely singular continuous spectrum. copyright 1997 American Institute of Physics

The changes of the interspace angle after anterior correction and instrumentation in adolescent idiopathic scoliosis patients

Directory of Open Access Journals (Sweden)

Fei Qi

2007-10-01

Full Text Available Abstract Background In idiopathic scoliosis patients, after anterior spinal fusion and instrumentation, the discs (interspace angle between the lowest instrumented vertebra (LIV and the next caudal vertebra became more wedged. We reviewed these patients and analyzed the changes of the angle. Methods By reviewing the medical records and roentgenograms of adolescent idiopathic scoliosis patients underwent anterior spinal fusion and instrumentation, Cobb angle of the curve, correction rate, coronal balance, LIV rotation, interspace angle were measured and analyzed. Results There were total 30 patients included. The mean coronal Cobb angle of the main curve (thoracolumbar/lumbar curve before and after surgery were 48.9° and 11.7°, respectively, with an average correction rate of 76.1%. The average rotation of LIV before surgery was 2.1 degree, and was improved to 1.2 degree after surgery. The interspace angle before surgery, on convex side-bending films, after surgery, at final follow up were 3.2°, -2.3°, 1.8° and 4.9°, respectively. The difference between the interspace angle after surgery and that preoperatively was not significant (P = 0.261, while the interspace angle at final follow-up became larger than that after surgery, and the difference was significant(P = 0.012. The interspace angle after surgery was correlated with that on convex side-bending films (r = 0.418, P = 0.022, and the interspace angle at final follow-up was correlated with that after surgery (r = 0.625, P = 0.000. There was significant correlation between the loss of the interspace angle and the loss of coronal Cobb angle of the main curve during follow-up(r = 0.483, P = 0.007. Conclusion The interspace angle could be improved after anterior correction and instrumentation surgery, but it became larger during follow-up. The loss of the interspace angle was correlated with the loss of coronal Cobb angle of the main curve during follow-up.

Interspace modification of titania-nanorod arrays for efficient mesoscopic perovskite solar cells

Energy Technology Data Exchange (ETDEWEB)

Chen, Peng; Jin, Zhixin; Wang, Yinglin, E-mail: wangyl100@nenu.edu.cn; Wang, Meiqi; Chen, Shixin; Zhang, Yang; Wang, Lingling; Zhang, Xintong, E-mail: xtzhang@nenu.edu.cn; Liu, Yichun, E-mail: ycliu@nenu.edu.cn

2017-04-30

Highlights: • The fabrication of perovskite solar cells utilizing TiO{sub 2} NR arrays. • Investigation of the interspace effect of TiO{sub 2} NR on perovskite layer. • Understanding of the balance between perovskite capping layer and pore filling. - Abstract: Morphology of electron transport layers (ETLs) has an important influence on the device architecture and electronic processes of mesostructured solar cells. In this work, we thoroughly investigated the effect of the interspace of TiO{sub 2} nanorod (NR) arrays on the photovoltaic performance of the perovskite solar cells (PSCs). Along with the interspace in TiO{sub 2}-NR arrays increasing, the thickness as well as the crystal size of perovskite capping layer are reduced accordingly, and the filling of perovskite in the channel becomes incomplete. Electrochemical impedance spectroscopy measurements reveal that this variation of perovskite absorber layer, induced by interspace of TiO{sub 2} NR arrays, causes the change of charge recombination process at the TiO{sub 2}/perovskite interface, suggesting that a balance between capping layer and the perovskite filling is critical to obtain high charge collection efficiency of PSCs. A power conversion efficiency of 10.3% could be achieved through careful optimization of interspace in TiO{sub 2}-NR arrays. Our research will shed light on the morphology control of ETLs with 1D structure for heterojunction solar cells fabricated by solution-deposited method.

Interspace modification of titania-nanorod arrays for efficient mesoscopic perovskite solar cells

Science.gov (United States)

Chen, Peng; Jin, Zhixin; Wang, Yinglin; Wang, Meiqi; Chen, Shixin; Zhang, Yang; Wang, Lingling; Zhang, Xintong; Liu, Yichun

2017-04-01

Morphology of electron transport layers (ETLs) has an important influence on the device architecture and electronic processes of mesostructured solar cells. In this work, we thoroughly investigated the effect of the interspace of TiO2 nanorod (NR) arrays on the photovoltaic performance of the perovskite solar cells (PSCs). Along with the interspace in TiO2-NR arrays increasing, the thickness as well as the crystal size of perovskite capping layer are reduced accordingly, and the filling of perovskite in the channel becomes incomplete. Electrochemical impedance spectroscopy measurements reveal that this variation of perovskite absorber layer, induced by interspace of TiO2 NR arrays, causes the change of charge recombination process at the TiO2/perovskite interface, suggesting that a balance between capping layer and the perovskite filling is critical to obtain high charge collection efficiency of PSCs. A power conversion efficiency of 10.3% could be achieved through careful optimization of interspace in TiO2-NR arrays. Our research will shed light on the morphology control of ETLs with 1D structure for heterojunction solar cells fabricated by solution-deposited method.

Mre11-Sae2 and RPA Collaborate to Prevent Palindromic Gene Amplification.

Science.gov (United States)

Deng, Sarah K; Yin, Yi; Petes, Thomas D; Symington, Lorraine S

2015-11-05

Foldback priming at DNA double-stranded breaks is one mechanism proposed to initiate palindromic gene amplification, a common feature of cancer cells. Here, we show that small (5-9 bp) inverted repeats drive the formation of large palindromic duplications, the major class of chromosomal rearrangements recovered from yeast cells lacking Sae2 or the Mre11 nuclease. RPA dysfunction increased the frequency of palindromic duplications in Sae2 or Mre11 nuclease-deficient cells by ∼ 1,000-fold, consistent with intra-strand annealing to create a hairpin-capped chromosome that is subsequently replicated to form a dicentric isochromosome. The palindromic duplications were frequently associated with duplication of a second chromosome region bounded by a repeated sequence and a telomere, suggesting the dicentric chromosome breaks and repairs by recombination between dispersed repeats to acquire a telomere. We propose secondary structures within single-stranded DNA are potent instigators of genome instability, and RPA and Mre11-Sae2 play important roles in preventing their formation and propagation, respectively. Copyright © 2015 Elsevier Inc. All rights reserved.

CRISPR-Cas9: a promising tool for gene editing on induced pluripotent stem cells.

Science.gov (United States)

Kim, Eun Ji; Kang, Ki Ho; Ju, Ji Hyeon

2017-01-01

Recent advances in genome editing with programmable nucleases have opened up new avenues for multiple applications, from basic research to clinical therapy. The ease of use of the technology-and particularly clustered regularly interspaced short palindromic repeats (CRISPR)-will allow us to improve our understanding of genomic variation in disease processes via cellular and animal models. Here, we highlight the progress made in correcting gene mutations in monogenic hereditary disorders and discuss various CRISPR-associated applications, such as cancer research, synthetic biology, and gene therapy using induced pluripotent stem cells. The challenges, ethical issues, and future prospects of CRISPR-based systems for human research are also discussed.

Applications of Engineered DNA-Binding Molecules Such as TAL Proteins and the CRISPR/Cas System in Biology Research

Directory of Open Access Journals (Sweden)

Toshitsugu Fujita

2015-09-01

Full Text Available Engineered DNA-binding molecules such as transcription activator-like effector (TAL or TALE proteins and the clustered regularly interspaced short palindromic repeats (CRISPR and CRISPR-associated proteins (Cas (CRISPR/Cas system have been used extensively for genome editing in cells of various types and species. The sequence-specific DNA-binding activities of these engineered DNA-binding molecules can also be utilized for other purposes, such as transcriptional activation, transcriptional repression, chromatin modification, visualization of genomic regions, and isolation of chromatin in a locus-specific manner. In this review, we describe applications of these engineered DNA-binding molecules for biological purposes other than genome editing.

Control of gene expression by CRISPR-Cas systems

Science.gov (United States)

2013-01-01

Clustered regularly interspaced short palindromic repeats (CRISPR) loci and their associated cas (CRISPR-associated) genes provide adaptive immunity against viruses (phages) and other mobile genetic elements in bacteria and archaea. While most of the early work has largely been dominated by examples of CRISPR-Cas systems directing the cleavage of phage or plasmid DNA, recent studies have revealed a more complex landscape where CRISPR-Cas loci might be involved in gene regulation. In this review, we summarize the role of these loci in the regulation of gene expression as well as the recent development of synthetic gene regulation using engineered CRISPR-Cas systems. PMID:24273648

CRISPR-Cas9: a promising tool for gene editing on induced pluripotent stem cells

Science.gov (United States)

Kim, Eun Ji; Kang, Ki Ho; Ju, Ji Hyeon

2017-01-01

Recent advances in genome editing with programmable nucleases have opened up new avenues for multiple applications, from basic research to clinical therapy. The ease of use of the technology—and particularly clustered regularly interspaced short palindromic repeats (CRISPR)—will allow us to improve our understanding of genomic variation in disease processes via cellular and animal models. Here, we highlight the progress made in correcting gene mutations in monogenic hereditary disorders and discuss various CRISPR-associated applications, such as cancer research, synthetic biology, and gene therapy using induced pluripotent stem cells. The challenges, ethical issues, and future prospects of CRISPR-based systems for human research are also discussed. PMID:28049282

Palindromic widths of nilpotent and wreath products

Indian Academy of Sciences (India)

Palindromic width; commutator width; wreath products; nilpotent product. 2000 Mathematics ... An algorithm of the computation of the commutator length in free non-abelian .... It is clear that A(1)B = A × B is the direct sum. Let us list some ...

Large palindromes in the lambda phage genome are preserved in a rec/sup +/ host by inhibiting lambda DNA replication

Energy Technology Data Exchange (ETDEWEB)

Shurvinton, C.E.; Stahl, M.M.; Stahl, F.W.

1987-03-01

A large palindrome carried by phage lambda has been shown to prevent growth of the phage on a rec/sup +/ strain of Escherichia coli. The phage do form plaques on recBC sbcB strains, but the palindrome is not stable - deletions that either destroy the palindrome or diminish its size overgrow the original engineered palindrome-containing phage. The authors have prepared stocks of lambda carrying a palindrome that is 2 x 4200 base pairs long. lambda phage were density labeled by UV induction of lysogens grown in minimal medium containing (/sup 13/C) glucose and /sup 15/NH/sub 4/Cl. These phage stocks are produced by induction of a lysogen in which the two halves of the palindrome are stored at opposite ends of the prophage and are of sufficient titer (10/sup 9/ phage per ml) to enable one-step growth experiments with replication-blocked phage. They find that the large palindrome as well as a lesser palindrome of 2 x 265 base pairs are recovered intact among particles carrying unreplicated chromosomes following such an infection of a rec/sup +/ host. they propose that DNA replication drives the extrusion of palindromic sequences in vivo, forming secondary structures that are substrates for the recBC and sbcB gene products.

Competition for phosphorus: differential uptake from dual-isotope-labeled soil interspaces between shrub and grass

International Nuclear Information System (INIS)

Caldwell, M.M.; Eissenstat, D.M.; Richards, J.H.; Allen, M.F.

1985-01-01

Two species of Agropyron grass differed strikingly in their capacity to compete for phosphate in soil interspaces shared with a common competitor, the sagebrush Artemisia tridentata. Of the total phosphorus-32 and -33 absorbed by Artemisia, 86% was from the interspace shared with Agropyron spicatum and only 14% from that shared with Agropyron desertorum. Actively absorbing mycorrhizal roots of Agropyron and Artemisia were present in both interspaces, where competition for the labeled phosphate occurred. The results have important implications about the way in which plants compete for resources below ground in both natural plant communities and agricultural intercropping systems

Bacteriophages of Yersinia pestis.

Science.gov (United States)

Zhao, Xiangna; Skurnik, Mikael

2016-01-01

Bacteriophage play many varied roles in microbial ecology and evolution. This chapter collates a vast body of knowledge and expertise on Yersinia pestis phages, including the history of their isolation and classical methods for their isolation and identification. The genomic diversity of Y. pestis phage and bacteriophage islands in the Y. pestis genome are also discussed because all phage research represents a branch of genetics. In addition, our knowledge of the receptors that are recognized by Y. pestis phage, advances in phage therapy for Y. pestis infections, the application of phage in the detection of Y. pestis, and clustered regularly interspaced short palindromic repeats (CRISPRs) sequences of Y. pestis from prophage DNA are all reviewed here.

CRISPR-Cas9 System as a Versatile Tool for Genome Engineering in Human Cells

Directory of Open Access Journals (Sweden)

Xuelian Wang

2016-01-01

Full Text Available Targeted nucleases are influential instruments for intervening in genome revision with great accuracy. RNA-guided Cas9 nucleases produced from clustered regularly interspaced short palindromic repeats (CRISPR-Cas systems have noticeably altered the means to modify the genomes of distinct organisms. They can be notably used to facilitate effective genome manipulation in eukaryotic cells by clearly detailing a 20-nt targeting sequence inside its directed RNA. We discuss the most recent advancements in the molecular basis of the type II CRISPR/Cas system and encapsulate applications and elements affecting its use in human cells. We also propose possible applications covering its uses ranging from basic science to implementation in the clinic.

Bacteria vs. bacteriophages: parallel evolution of immune arsenals

Directory of Open Access Journals (Sweden)

Muhammad Abu Bakr Shabbir

2016-08-01

Full Text Available Bacteriophages are the most common entities on earth and represent a constant challenge to bacterial populations. To fend off bacteriophage infection, bacteria evolved immune systems to avert phage adsorption and block invader DNA entry. They developed restriction-modification systems and mechanisms to abort infection and interfere with virion assembly, as well as newly recognized clustered regularly interspaced short palindromic repeats (CRISPR. In response to bacterial immune systems, bacteriophages synchronously evolved resistance mechanisms, such as the anti-CRISPR systems to counterattack bacterial CRISPR-cas systems, in a continuing evolutionary arms race between virus and host. In turn, it is fundamental to the survival of the bacterial cell to evolve a system to combat bacteriophage immune strategies.

Unravelling the structural and mechanistic basis of CRISPR-Cas systems.

Science.gov (United States)

van der Oost, John; Westra, Edze R; Jackson, Ryan N; Wiedenheft, Blake

2014-07-01

Bacteria and archaea have evolved sophisticated adaptive immune systems, known as CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems, which target and inactivate invading viruses and plasmids. Immunity is acquired by integrating short fragments of foreign DNA into CRISPR loci, and following transcription and processing of these loci, the CRISPR RNAs (crRNAs) guide the Cas proteins to complementary invading nucleic acid, which results in target interference. In this Review, we summarize the recent structural and biochemical insights that have been gained for the three major types of CRISPR-Cas systems, which together provide a detailed molecular understanding of the unique and conserved mechanisms of RNA-guided adaptive immunity in bacteria and archaea.

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape.

Science.gov (United States)

Ferreira, Raphael; David, Florian; Nielsen, Jens

2018-01-24

Clustered regularly interspaced short palindromic repeats (CRISPR) is poised to become one of the key scientific discoveries of the twenty-first century. Originating from prokaryotic and archaeal immune systems to counter phage invasions, CRISPR-based applications have been tailored for manipulating a broad range of living organisms. From the different elucidated types of CRISPR mechanisms, the type II system adapted from Streptococcus pyogenes has been the most exploited as a tool for genome engineering and gene regulation. In this review, we describe the different applications of CRISPR/Cas9 technology in the industrial biotechnology field. Next, we detail the current status of the patent landscape, highlighting its exploitation through different companies, and conclude with future perspectives of this technology.

CRISPR-Cas Targeting of Host Genes as an Antiviral Strategy.

Science.gov (United States)

Chen, Shuliang; Yu, Xiao; Guo, Deyin

2018-01-16

Currently, a new gene editing tool-the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated (Cas) system-is becoming a promising approach for genetic manipulation at the genomic level. This simple method, originating from the adaptive immune defense system in prokaryotes, has been developed and applied to antiviral research in humans. Based on the characteristics of virus-host interactions and the basic rules of nucleic acid cleavage or gene activation of the CRISPR-Cas system, it can be used to target both the virus genome and host factors to clear viral reservoirs and prohibit virus infection or replication. Here, we summarize recent progress of the CRISPR-Cas technology in editing host genes as an antiviral strategy.

Unravelling the structural and mechanistic basis of CRISPR–Cas systems

Science.gov (United States)

van der Oost, John; Westra, Edze R.; Jackson, Ryan N.; Wiedenheft, Blake

2014-01-01

Bacteria and archaea have evolved sophisticated adaptive immune systems, known as CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) systems, which target and inactivate invading viruses and plasmids. Immunity is acquired by integrating short fragments of foreign DNA into CRISPR loci, and following transcription and processing of these loci, the CRISPR RNAs (crRNAs) guide the Cas proteins to complementary invading nucleic acid, which results in target interference. In this Review, we summarize the recent structural and biochemical insights that have been gained for the three major types of CRISPR–Cas systems, which together provide a detailed molecular understanding of the unique and conserved mechanisms of RNA-guided adaptive immunity in bacteria and archaea. PMID:24909109

Heterogeneous diversity of spacers within CRISPR

Science.gov (United States)

Deem, Michael; He, Jiankui

2011-03-01

Clustered regularly interspaced short palindromic repeats (CRISPR) in bacterial and archaeal DNA have recently been shown to be a new type of anti-viral immune system in these organisms. We here study the diversity of spacers in CRISPR under selective pressure. We propose a population dynamics model that explains the biological observation that the leader-proximal end of CRISPR is more diversified and the leader-distal end of CRISPR is more conserved. This result is shown to be in agreement with recent experiments. Our results show that the CRISPR spacer structure is influenced by and provides a record of the viral challenges that bacteria face. 1) J. He and M. W. Deem, Phys. Rev. Lett. 105 (2010) 128102

New insights into the nanometer-scaled cell-surface interspace by cell-sensor measurements

International Nuclear Information System (INIS)

Lehmann, Mirko; Baumann, Werner

2005-01-01

The culture of adherent cells on solid surfaces is an established in vitro method, and the adhesion process of a cell is considered as an important trigger for many cellular processes (e.g., polarity and tumor genesis). However, not all of the eliciting biochemical or biophysical reactions are yet understood. Interestingly, there are not much experimental data about the impact that the interspace between an adherent cell and the (solid) substrate has on the cell's behavior. This interspace is mainly built by the basolateral side of epithelial cells and the substrate. This paper gives some new results of non-invasive and non-optical measurements in the interspace. The measurements were made with silicon cell-sensor hybrids. Measurements of acidification, adhesion, and respiration are analyzed in view of the situation in the interspace. The results show that, in general, the release of an ion or molecule on the basolateral side can have much more influence on the biophysical situation than a release of an ion or molecule on the apical side. In particular, the apical acidification (i.e., amount of extruded protons) of, e.g., epithelial tumor cells is several orders of magnitude higher than the basolateral acidification. These experimental results are a simple consequence of the fact that the basolateral volume of the interspace is several orders of magnitudes smaller than the apical volume. These results have the following consequences for the cell adhesion:a)static situation: if a cell is already adhered to a solid substrate, the basolateral and apical release and uptake of molecules have to be considered in a very differentiated way; b)dynamic situation: if the cell is adhering to the substrate, the then built basolateral side changes in a much stronger way than the apical side. This effect is here discussed as a possible eliciting and general mechanism for essential intracellular changes

CRISPR-Cas type I-A Cascade complex couples viral infection surveillance to host transcriptional regulation in the dependence of Csa3b

Science.gov (United States)

He, Fei; Vestergaard, Gisle; Peng, Wenfang; She, Qunxin

2017-01-01

Abstract CRISPR-Cas (clustered regularly interspaced short palindromic repeats and the associated genes) constitute adaptive immune systems in bacteria and archaea and they provide sequence specific immunity against foreign nucleic acids. CRISPR-Cas systems are activated by viral infection. However, little is known about how CRISPR-Cas systems are activated in response to viral infection or how their expression is controlled in the absence of viral infection. Here, we demonstrate that both the transcriptional regulator Csa3b, and the type I-A interference complex Cascade, are required to transcriptionally repress the interference gene cassette in the archaeon Sulfolobus. Csa3b binds to two palindromic repeat sites in the promoter region of the cassette and facilitates binding of the Cascade to the promoter region. Upon viral infection, loading of Cascade complexes onto crRNA-matching protospacers leads to relief of the transcriptional repression. Our data demonstrate a mechanism coupling CRISPR-Cas surveillance of protospacers to transcriptional regulation of the interference gene cassette thereby allowing a fast response to viral infection. PMID:27980065

Myocardial Infarction in a Young Female with Palindromic Rheumatism: A Consequence of Negative Remodeling

Directory of Open Access Journals (Sweden)

Timothy R. Larsen

2012-01-01

Full Text Available Palindromic rheumatism is a rare disease associated with systemic inflammation. Negative or constrictive coronary artery remodeling is typically not seen until the 7th or 8th decade of life. We report a case of a young female with palindromic rheumatism who suffered a non-ST segment elevation myocardial infarction secondary to a flow-limiting lesion that demonstrated negative remodeling by intravascular ultrasound (IVUS.

Bacterial CRISPR Regions: General Features and their Potential for Epidemiological Molecular Typing Studies.

Science.gov (United States)

Karimi, Zahra; Ahmadi, Ali; Najafi, Ali; Ranjbar, Reza

2018-01-01

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci as novel and applicable regions in prokaryotic genomes have gained great attraction in the post genomics era. These unique regions are diverse in number and sequence composition in different pathogenic bacteria and thereby can be a suitable candidate for molecular epidemiology and genotyping studies. Results:Furthermore, the arrayed structure of CRISPR loci (several unique repeats spaced with the variable sequence) and associated cas genes act as an active prokaryotic immune system against viral replication and conjugative elements. This property can be used as a tool for RNA editing in bioengineering studies. The aim of this review was to survey some details about the history, nature, and potential applications of CRISPR arrays in both genetic engineering and bacterial genotyping studies.

The Future of CRISPR Applications in the Lab, the Clinic and Society.

Science.gov (United States)

Hough, Soren H; Ajetunmobi, Ayokunmi

2017-01-01

CRISPR (clustered regularly interspaced short palindromic repeats) has emerged as one of the premiere biological tools of the century. Even more so than older genome editing techniques such as TALENs and ZFNs, CRISPR provides speed and ease-of-use heretofore unheard of in agriculture, the environment and human health. The ability to map the function of virtually every component of the genome in a scalable, multiplexed manner is unprecedented. Once those regions have been explored, CRISPR also presents an opportunity to take advantage of endogenous cellular repair pathways to change and precisely edit the genome [1-3]. In the case of human health, CRISPR operates as both a tool of discovery and a solution to fundamental problems behind disease and undesirable mutations.

Genetic and epigenetic control of gene expression by CRISPR–Cas systems

Science.gov (United States)

Lo, Albert; Qi, Lei

2017-01-01

The discovery and adaption of bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems has revolutionized the way researchers edit genomes. Engineering of catalytically inactivated Cas variants (nuclease-deficient or nuclease-deactivated [dCas]) combined with transcriptional repressors, activators, or epigenetic modifiers enable sequence-specific regulation of gene expression and chromatin state. These CRISPR–Cas-based technologies have contributed to the rapid development of disease models and functional genomics screening approaches, which can facilitate genetic target identification and drug discovery. In this short review, we will cover recent advances of CRISPR–dCas9 systems and their use for transcriptional repression and activation, epigenome editing, and engineered synthetic circuits for complex control of the mammalian genome. PMID:28649363

CRISPR-cas loci profiling of Cronobacter sakazakii pathovars.

Science.gov (United States)

Ogrodzki, Pauline; Forsythe, Stephen James

2016-12-01

Cronobacter sakazakii sequence types 1, 4, 8 and 12 are associated with outbreaks of neonatal meningitis and necrotizing enterocolitis infections. However clonality results in strains which are indistinguishable using conventional methods. This study investigated the use of clustered regularly interspaced short palindromic repeats (CRISPR)-cas loci profiling for epidemiological investigations. Seventy whole genomes of C. sakazakii strains from four clonal complexes which were widely distributed temporally, geographically and origin of source were profiled. All strains encoded the same type I-E subtype CRISPR-cas system with a total of 12 different CRISPR spacer arrays. This study demonstrated the greater discriminatory power of CRISPR spacer array profiling compared with multilocus sequence typing, which will be of use in source attribution during Cronobacter outbreak investigations.

Efficient engineering of a bacteriophage genome using the type I-E CRISPR-Cas system.

Science.gov (United States)

Kiro, Ruth; Shitrit, Dror; Qimron, Udi

2014-01-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system has recently been used to engineer genomes of various organisms, but surprisingly, not those of bacteriophages (phages). Here we present a method to genetically engineer the Escherichia coli phage T7 using the type I-E CRISPR-Cas system. T7 phage genome is edited by homologous recombination with a DNA sequence flanked by sequences homologous to the desired location. Non-edited genomes are targeted by the CRISPR-Cas system, thus enabling isolation of the desired recombinant phages. This method broadens CRISPR Cas-based editing to phages and uses a CRISPR-Cas type other than type II. The method may be adjusted to genetically engineer any bacteriophage genome.

Genome Engineering with TALE and CRISPR Systems in Neuroscience.

Science.gov (United States)

Lee, Han B; Sundberg, Brynn N; Sigafoos, Ashley N; Clark, Karl J

2016-01-01

Recent advancement in genome engineering technology is changing the landscape of biological research and providing neuroscientists with an opportunity to develop new methodologies to ask critical research questions. This advancement is highlighted by the increased use of programmable DNA-binding agents (PDBAs) such as transcription activator-like effector (TALE) and RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) systems. These PDBAs fused or co-expressed with various effector domains allow precise modification of genomic sequences and gene expression levels. These technologies mirror and extend beyond classic gene targeting methods contributing to the development of novel tools for basic and clinical neuroscience. In this Review, we discuss the recent development in genome engineering and potential applications of this technology in the field of neuroscience.

CRISPR-based immune systems of the Sulfolobales: complexity and diversity

DEFF Research Database (Denmark)

Garrett, Roger Antony; Shah, Shiraz Ali; Vestergaard, Gisle Alberg

2011-01-01

CRISPR (cluster of regularly interspaced palindromic repeats)/Cas and CRISPR/Cmr systems of Sulfolobus, targeting DNA and RNA respectively of invading viruses or plasmids are complex and diverse. We address their classification and functional diversity, and the wide sequence diversity of RAMP...... (repeat-associated mysterious protein)-motif containing proteins encoded in Cmr modules. Factors influencing maintenance of partially impaired CRISPR-based systems are discussed. The capacity for whole CRISPR transcripts to be generated despite the uptake of transcription signals within spacer sequences...... is considered. Targeting of protospacer regions of invading elements by Cas protein-crRNA (CRISPR RNA) complexes exhibit relatively low sequence stringency, but the integrity of protospacer-associated motifs appears to be important. Different mechanisms for circumventing or inactivating the immune systems...

CRISPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins.

Science.gov (United States)

Couvin, David; Bernheim, Aude; Toffano-Nioche, Claire; Touchon, Marie; Michalik, Juraj; Néron, Bertrand; C Rocha, Eduardo P; Vergnaud, Gilles; Gautheret, Daniel; Pourcel, Christine

2018-05-22

CRISPR (clustered regularly interspaced short palindromic repeats) arrays and their associated (Cas) proteins confer bacteria and archaea adaptive immunity against exogenous mobile genetic elements, such as phages or plasmids. CRISPRCasFinder allows the identification of both CRISPR arrays and Cas proteins. The program includes: (i) an improved CRISPR array detection tool facilitating expert validation based on a rating system, (ii) prediction of CRISPR orientation and (iii) a Cas protein detection and typing tool updated to match the latest classification scheme of these systems. CRISPRCasFinder can either be used online or as a standalone tool compatible with Linux operating system. All third-party software packages employed by the program are freely available. CRISPRCasFinder is available at https://crisprcas.i2bc.paris-saclay.fr.

High-efficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairs

DEFF Research Database (Denmark)

Duda, Katarzyna; Lonowski, Lindsey A; Kofoed-Nielsen, Michael

2014-01-01

Targeted endonucleases including zinc finger nucleases (ZFNs) and clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas9 are increasingly being used for genome editing in higher species. We therefore devised a broadly applicable and versatile method for increasing editing...... higher genome editing rates. For ZFNs, this approach, combined with delivery of donors as single-stranded oligodeoxynucleotides and nucleases as messenger ribonucleic acid, enabled high knockin efficiencies in demanding applications, including biallelic codon conversion frequencies reaching 30......-70% at high transfection efficiencies and ∼2% at low transfection efficiencies, simultaneous homozygous knockin mutation of two genes with ∼1.5% efficiency as well as generation of cell pools with almost complete codon conversion via three consecutive targeting and FACS events. Observed off-target effects...

Translation of CRISPR Genome Surgery to the Bedside for Retinal Diseases

Directory of Open Access Journals (Sweden)

Christine L. Xu

2018-05-01

Full Text Available In recent years, there has been accelerated growth of clustered regularly interspaced short palindromic repeats (CRISPR genome surgery techniques. Genome surgery holds promise for diseases for which a cure currently does not exist. In the field of ophthalmology, CRISPR offers possibilities for treating inherited retinal dystrophies. The retina has little regenerative potential, which makes treatment particularly difficult. For such conditions, CRISPR genome surgery methods have shown great potential for therapeutic applications in animal models of retinal dystrophies. Much anticipation surrounds the potential for CRISPR as a therapeutic, as clinical trials of ophthalmic genome surgery are expected to begin as early as 2018. This mini-review summarizes preclinical CRISPR applications in the retina and current CRISPR clinical trials.

Targeted Porcine Genome Engineering with TALENs

DEFF Research Database (Denmark)

Luo, Yonglun; Lin, Lin; Golas, Mariola Monika

2015-01-01

Genetically modified pigs are becoming an invaluable animal model for agricultural, pharmaceutical, and biomedical applications. Unlike traditional transgenesis, which is accomplished by randomly inserting an exogenous transgene cassette into the natural chromosomal context, targeted genome editing...... confers precisely editing (e.g., mutations or indels) or insertion of a functional transgenic cassette to user-designed loci. Techniques for targeted genome engineering are growing dramatically and include, e.g., zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs......), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems. These systems provide enormous potential applications. In this chapter, we review the use of TALENs for targeted genome editing with focus on their application in pigs. In addition, a brief protocol...

In-service inspection in the Superphenix 1 vessels interspace

International Nuclear Information System (INIS)

Asty, M.; Saglio, R.

1983-03-01

The design of Superphenix 1 reactor vessels allows their in-service inspection. A self-propelling engine, the MIR, has been concieved for this need: it can do a visual and ultrasonic inspection. The MIR can move in the whole vessels interspace. The operating conditions are specified and the principle characteristics of the MIR engine are presented [fr

An algorithm to find all palindromic sequences in proteins

Indian Academy of Sciences (India)

2013-01-20

Jan 20, 2013 ... 1976; Karrer and Gall 1976; Vogt and Braun 1976) and (iii) in the formation of hairpin loops in the newly transcribed RNA. Palindromic sequences are observed in various classes of proteins like histones (Cheng et al. 1989), prion proteins (Sulkowski 1992; Kazim 1993),. DNA-binding proteins (Suzuki 1992; ...

Functional Analysis of Porphyromonas gingivalis W83 CRISPR-Cas Systems.

Science.gov (United States)

Burmistrz, Michał; Dudek, Bartosz; Staniec, Dominika; Rodriguez Martinez, Jose Ignacio; Bochtler, Matthias; Potempa, Jan; Pyrc, Krzysztof

2015-08-01

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) system provides prokaryotic cells with an adaptive and heritable immune response to foreign genetic elements, such as viruses, plasmids, and transposons. It is present in the majority of Archaea and almost half of species of Bacteria. Porphyromonas gingivalis is an important human pathogen that has been proven to be an etiological agent of periodontitis and has been linked to systemic conditions, such as rheumatoid arthritis and cardiovascular disease. At least 95% of clinical strains of P. gingivalis carry CRISPR arrays, suggesting that these arrays play an important function in vivo. Here we show that all four CRISPR arrays present in the P. gingivalis W83 genome are transcribed. For one of the arrays, we demonstrate in vivo activity against double-stranded DNA constructs containing protospacer sequences accompanied at the 3' end by an NGG protospacer-adjacent motif (PAM). Most of the 44 spacers present in the genome of P. gingivalis W83 share no significant similarity with any known sequences, although 4 spacers are similar to sequences from bacteria found in the oral cavity and the gastrointestinal tract. Four spacers match genomic sequences of the host; however, none of these is flanked at its 3' terminus by the appropriate PAM element. The CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) system is a unique system that provides prokaryotic cells with an adaptive and heritable immunity. In this report, we show that the CRISPR-Cas system of P. gingivalis, an important human pathogen associated with periodontitis and possibly also other conditions, such as rheumatoid arthritis and cardiovascular disease, is active and provides protection from foreign genetic elements. Importantly, the data presented here may be useful for better understanding the communication between cells in larger bacterial communities and

CRISPR-Cas: biology, mechanisms and relevance

Science.gov (United States)

Hille, Frank

2016-01-01

Prokaryotes have evolved several defence mechanisms to protect themselves from viral predators. Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) display a prokaryotic adaptive immune system that memorizes previous infections by integrating short sequences of invading genomes—termed spacers—into the CRISPR locus. The spacers interspaced with repeats are expressed as small guide CRISPR RNAs (crRNAs) that are employed by Cas proteins to target invaders sequence-specifically upon a reoccurring infection. The ability of the minimal CRISPR-Cas9 system to target DNA sequences using programmable RNAs has opened new avenues in genome editing in a broad range of cells and organisms with high potential in therapeutical applications. While numerous scientific studies have shed light on the biochemical processes behind CRISPR-Cas systems, several aspects of the immunity steps, however, still lack sufficient understanding. This review summarizes major discoveries in the CRISPR-Cas field, discusses the role of CRISPR-Cas in prokaryotic immunity and other physiological properties, and describes applications of the system as a DNA editing technology and antimicrobial agent. This article is part of the themed issue ‘The new bacteriology’. PMID:27672148

CRISPR-Cas type I-A Cascade complex couples viral infection surveillance to host transcriptional regulation in the dependence of Csa3b.

Science.gov (United States)

He, Fei; Vestergaard, Gisle; Peng, Wenfang; She, Qunxin; Peng, Xu

2017-02-28

CRISPR-Cas (clustered regularly interspaced short palindromic repeats and the associated genes) constitute adaptive immune systems in bacteria and archaea and they provide sequence specific immunity against foreign nucleic acids. CRISPR-Cas systems are activated by viral infection. However, little is known about how CRISPR-Cas systems are activated in response to viral infection or how their expression is controlled in the absence of viral infection. Here, we demonstrate that both the transcriptional regulator Csa3b, and the type I-A interference complex Cascade, are required to transcriptionally repress the interference gene cassette in the archaeon Sulfolobus. Csa3b binds to two palindromic repeat sites in the promoter region of the cassette and facilitates binding of the Cascade to the promoter region. Upon viral infection, loading of Cascade complexes onto crRNA-matching protospacers leads to relief of the transcriptional repression. Our data demonstrate a mechanism coupling CRISPR-Cas surveillance of protospacers to transcriptional regulation of the interference gene cassette thereby allowing a fast response to viral infection. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Applications of CRISPR/Cas9 in retinal degenerative diseases

Science.gov (United States)

Peng, Ying-Qian; Tang, Luo-Sheng; Yoshida, Shigeo; Zhou, Ye-Di

2017-01-01

Gene therapy is a potentially effective treatment for retinal degenerative diseases. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been developed as a new genome-editing tool in ophthalmic studies. Recent advances in researches showed that CRISPR/Cas9 has been applied in generating animal models as well as gene therapy in vivo of retinitis pigmentosa (RP) and leber congenital amaurosis (LCA). It has also been shown as a potential attempt for clinic by combining with other technologies such as adeno-associated virus (AAV) and induced pluripotent stem cells (iPSCs). In this review, we highlight the main points of further prospect of using CRISPR/Cas9 in targeting retinal degeneration. We also emphasize the potential applications of this technique in treating retinal degenerative diseases. PMID:28503441

Generation of an ICF syndrome model by efficient genome editing of human induced pluripotent stem cells using the CRISPR system.

Science.gov (United States)

Horii, Takuro; Tamura, Daiki; Morita, Sumiyo; Kimura, Mika; Hatada, Izuho

2013-09-30

Genome manipulation of human induced pluripotent stem (iPS) cells is essential to achieve their full potential as tools for regenerative medicine. To date, however, gene targeting in human pluripotent stem cells (hPSCs) has proven to be extremely difficult. Recently, an efficient genome manipulation technology using the RNA-guided DNase Cas9, the clustered regularly interspaced short palindromic repeats (CRISPR) system, has been developed. Here we report the efficient generation of an iPS cell model for immunodeficiency, centromeric region instability, facial anomalies syndrome (ICF) syndrome using the CRISPR system. We obtained iPS cells with mutations in both alleles of DNA methyltransferase 3B (DNMT3B) in 63% of transfected clones. Our data suggest that the CRISPR system is highly efficient and useful for genome engineering of human iPS cells.

[CRISPR/Cas system for genome editing in pluripotent stem cells].

Science.gov (United States)

Vasil'eva, E A; Melino, D; Barlev, N A

2015-01-01

Genome editing systems based on site-specific nucleases became very popular for genome editing in modern bioengineering. Human pluripotent stem cells provide a unique platform for genes function study, disease modeling, and drugs testing. Consequently, technology for fast, accurate and well controlled genome manipulation is required. CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRISPR-associated) system could be employed for these purposes. This system is based on site-specific programmable nuclease Cas9. Numerous advantages of the CRISPR/Cas system and its successful application to human stem cells provide wide opportunities for genome therapy and regeneration medicine. In this publication, we describe and compare the main genome editing systems based on site-specific programmable nucleases and discuss opportunities and perspectives of the CRISPR/Cas system for application to pluripotent stem cells.

[Efficient genome editing in human pluripotent stem cells through CRISPR/Cas9].

Science.gov (United States)

Liu, Gai-gai; Li, Shuang; Wei, Yu-da; Zhang, Yong-xian; Ding, Qiu-rong

2015-11-01

The RNA-guided CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas9 nuclease has offered a new platform for genome editing with high efficiency. Here, we report the use of CRISPR/Cas9 technology to target a specific genomic region in human pluripotent stem cells. We show that CRISPR/Cas9 can be used to disrupt a gene by introducing frameshift mutations to gene coding region; to knock in specific sequences (e.g. FLAG tag DNA sequence) to targeted genomic locus via homology directed repair; to induce large genomic deletion through dual-guide multiplex. Our results demonstrate the versatile application of CRISPR/Cas9 in stem cell genome editing, which can be widely utilized for functional studies of genes or genome loci in human pluripotent stem cells.

CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER) for Near-Perfect Selective Transformation

Science.gov (United States)

Rothschild, Lynn J.; Greenberg, Daniel T.; Takahashi, Jack R.; Thompson, Kirsten A.; Maheshwari, Akshay J.; Kent, Ryan E.; McCutcheon, Griffin; Shih, Joseph D.; Calvet, Charles; Devlin, Tyler D.;

Functional interrogation of non-coding DNA through CRISPR genome editing.

Science.gov (United States)

Canver, Matthew C; Bauer, Daniel E; Orkin, Stuart H

2017-05-15

Methodologies to interrogate non-coding regions have lagged behind coding regions despite comprising the vast majority of the genome. However, the rapid evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing has provided a multitude of novel techniques for laboratory investigation including significant contributions to the toolbox for studying non-coding DNA. CRISPR-mediated loss-of-function strategies rely on direct disruption of the underlying sequence or repression of transcription without modifying the targeted DNA sequence. CRISPR-mediated gain-of-function approaches similarly benefit from methods to alter the targeted sequence through integration of customized sequence into the genome as well as methods to activate transcription. Here we review CRISPR-based loss- and gain-of-function techniques for the interrogation of non-coding DNA. Copyright © 2017 Elsevier Inc. All rights reserved.

Genome Engineering with TALE and CRISPR Systems in Neuroscience

Science.gov (United States)

Lee, Han B.; Sundberg, Brynn N.; Sigafoos, Ashley N.; Clark, Karl J.

2016-01-01

Recent advancement in genome engineering technology is changing the landscape of biological research and providing neuroscientists with an opportunity to develop new methodologies to ask critical research questions. This advancement is highlighted by the increased use of programmable DNA-binding agents (PDBAs) such as transcription activator-like effector (TALE) and RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) systems. These PDBAs fused or co-expressed with various effector domains allow precise modification of genomic sequences and gene expression levels. These technologies mirror and extend beyond classic gene targeting methods contributing to the development of novel tools for basic and clinical neuroscience. In this Review, we discuss the recent development in genome engineering and potential applications of this technology in the field of neuroscience. PMID:27092173

SURVIVAL AND EVOLUTION OF CRISPR-CAS SYSTEM IN PROKARYOTES AND ITS APPLICATIONS

Directory of Open Access Journals (Sweden)

Muhammad Abu Bakr Shabbir

2016-09-01

Full Text Available Prokaryotes have developed numerous innate immune mechanisms in order to fend off bacteriophage or plasmid attack. One of these immune systems is Clustered regularly interspaced short palindromic repeats (CRISPR. CRISPR associated proteins play a key role in survival of prokaryotes against invaders, as these systems cleave DNA of foreign genetic elements. Beyond providing immunity, these systems have significant impact in altering the bacterial physiology in term of its virulence and pathogenicity, as well as evolution. Also, due to their diverse nature of functionality, cas9 endoribonuclease can be easily reprogrammed with the help of guide RNAs, showing unprecedented potential and significance for gene editing in treating genetic diseases. Here, we also discuss the use of NgAgo-gDNA system in genome editing of human cells.

Survival and Evolution of CRISPR–Cas System in Prokaryotes and Its Applications

Science.gov (United States)

Shabbir, Muhammad Abu Bakr; Hao, Haihong; Shabbir, Muhammad Zubair; Hussain, Hafiz Iftikhar; Iqbal, Zahid; Ahmed, Saeed; Sattar, Adeel; Iqbal, Mujahid; Li, Jun; Yuan, Zonghui

2016-01-01

Prokaryotes have developed numerous innate immune mechanisms in order to fend off bacteriophage or plasmid attack. One of these immune systems is clustered regularly interspaced short palindromic repeats (CRISPR). CRISPR-associated proteins play a key role in survival of prokaryotes against invaders, as these systems cleave DNA of foreign genetic elements. Beyond providing immunity, these systems have significant impact in altering the bacterial physiology in term of its virulence and pathogenicity, as well as evolution. Also, due to their diverse nature of functionality, cas9 endoribonuclease can be easily reprogrammed with the help of guide RNAs, showing unprecedented potential and significance for gene editing in treating genetic diseases. Here, we also discuss the use of NgAgo–gDNA system in genome editing of human cells. PMID:27725818

Exploiting CRISPR/Cas: Interference Mechanisms and Applications

Directory of Open Access Journals (Sweden)

André Plagens

2013-07-01

Full Text Available The discovery of biological concepts can often provide a framework for the development of novel molecular tools, which can help us to further understand and manipulate life. One recent example is the elucidation of the prokaryotic adaptive immune system, clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR-associated (Cas that protects bacteria and archaea against viruses or conjugative plasmids. The immunity is based on small RNA molecules that are incorporated into versatile multi-domain proteins or protein complexes and specifically target viral nucleic acids via base complementarity. CRISPR/Cas interference machines are utilized to develop novel genome editing tools for different organisms. Here, we will review the latest progress in the elucidation and application of prokaryotic CRISPR/Cas systems and discuss possible future approaches to exploit the potential of these interference machineries.

From hacking the human genome to editing organs.

Science.gov (United States)

Tobita, Takamasa; Guzman-Lepe, Jorge; Collin de l'Hortet, Alexandra

2015-01-01

In the recent decades, human genome engineering has been one of the major interesting research subjects, essentially because it raises new possibilities for personalized medicine and biotechnologies. With the development of engineered nucleases such as the Zinc Finger Nucleases (ZFNs), the Transcription activator-like effector nucleases (TALENs) and more recently the Clustered Regularly Interspaced short Palindromic Repeats (CRISPR), the field of human genome edition has evolved very rapidly. Every new genetic tool is broadening the scope of applications on human tissues, even before we can completely master each of these tools. In this review, we will present the recent advances regarding human genome edition tools, we will discuss the numerous implications they have in research and medicine, and we will mention the limits and concerns about such technologies.

SMV1 virus-induced CRISPR spacer acquisition from the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2

DEFF Research Database (Denmark)

Erdmann, Susanne; Shah, Shiraz Ali; Garrett, Roger Antony

2013-01-01

Organisms of the crenarchaeal order Sulfolobales carry complex CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune systems. These systems are modular and show extensive structural and functional diversity, especially in their interference complexes. The primary...... targets are an exceptional range of diverse viruses, many of which propagate stably within cells and follow lytic life cycles without producing cell lysis. These properties are consistent with the difficulty of activating CRISPR spacer uptake in the laboratory, but appear to conflict with the high...... complexity and diversity of the CRISPR immune systems that are found among the Sulfolobales. In the present article, we re-examine the first successful induction of archaeal spacer acquisition in our laboratory that occurred exclusively for the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2...

Solution properties of the archaeal CRISPR DNA repeat-binding homeodomain protein Cbp2

DEFF Research Database (Denmark)

Kenchappa, Chandra; Heiðarsson, Pétur Orri; Kragelund, Birthe

2013-01-01

Clustered regularly interspaced short palindromic repeats (CRISPR) form the basis of diverse adaptive immune systems directed primarily against invading genetic elements of archaea and bacteria. Cbp1 of the crenarchaeal thermoacidophilic order Sulfolobales, carrying three imperfect repeats, binds...... specifically to CRISPR DNA repeats and has been implicated in facilitating production of long transcripts from CRISPR loci. Here, a second related class of CRISPR DNA repeat-binding protein, denoted Cbp2, is characterized that contains two imperfect repeats and is found amongst members of the crenarchaeal...... in facilitating high affinity DNA binding of Cbp2 by tethering the two domains. Structural studies on mutant proteins provide support for Cys(7) and Cys(28) enhancing high thermal stability of Cbp2(Hb) through disulphide bridge formation. Consistent with their proposed CRISPR transcriptional regulatory role, Cbp2...

Genetic Tools for Self-Organizing Culture of Mouse Embryonic Stem Cells via Small Regulatory RNA-Mediated Technologies, CRISPR/Cas9, and Inducible RNAi.

Science.gov (United States)

Takata, Nozomu; Sakakura, Eriko; Sakuma, Tetsushi; Yamamoto, Takashi

2017-01-01

Approaches to investigate gene functions in experimental biology are becoming more diverse and reliable. Furthermore, several kinds of tissues and organs that possess their original identities can be generated in petri dishes from stem cells including embryonic, adult and induced pluripotent stem cells. Researchers now have several choices of experimental methods and their combinations to analyze gene functions in various biological systems. Here, as an example we describe one of the better protocols, which combines three-dimensional embryonic stem cell culture with small regulatory RNA-mediated technologies, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), and inducible RNA interference (RNAi). This protocol allows investigation of genes of interest to better understand gene functions in target tissues (or organs) during in vitro development.

Exploiting CRISPR/Cas: Interference Mechanisms and Applications

Science.gov (United States)

Richter, Hagen; Randau, Lennart; Plagens, André

2013-01-01

The discovery of biological concepts can often provide a framework for the development of novel molecular tools, which can help us to further understand and manipulate life. One recent example is the elucidation of the prokaryotic adaptive immune system, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) that protects bacteria and archaea against viruses or conjugative plasmids. The immunity is based on small RNA molecules that are incorporated into versatile multi-domain proteins or protein complexes and specifically target viral nucleic acids via base complementarity. CRISPR/Cas interference machines are utilized to develop novel genome editing tools for different organisms. Here, we will review the latest progress in the elucidation and application of prokaryotic CRISPR/Cas systems and discuss possible future approaches to exploit the potential of these interference machineries. PMID:23857052

Antiviral Goes Viral: Harnessing CRISPR/Cas9 to Combat Viruses in Humans.

Science.gov (United States)

Soppe, Jasper Adriaan; Lebbink, Robert Jan

2017-10-01

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems are RNA-guided sequence-specific prokaryotic antiviral immune systems. In prokaryotes, small RNA molecules guide Cas effector endonucleases to invading foreign genetic elements in a sequence-dependent manner, resulting in DNA cleavage by the endonuclease upon target binding. A rewired CRISPR/Cas9 system can be used for targeted and precise genome editing in eukaryotic cells. CRISPR/Cas has also been harnessed to target human pathogenic viruses as a potential new antiviral strategy. Here, we review recent CRISPR/Cas9-based approaches to combat specific human viruses in humans and discuss challenges that need to be overcome before CRISPR/Cas9 may be used in the clinic as an antiviral strategy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Adaptation in CRISPR-Cas Systems.

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Sternberg, Samuel H; Richter, Hagen; Charpentier, Emmanuelle; Qimron, Udi

2016-03-17

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive immune system in prokaryotes. The system preserves memories of prior infections by integrating short segments of foreign DNA, termed spacers, into the CRISPR array in a process termed adaptation. During the past 3 years, significant progress has been made on the genetic requirements and molecular mechanisms of adaptation. Here we review these recent advances, with a focus on the experimental approaches that have been developed, the insights they generated, and a proposed mechanism for self- versus non-self-discrimination during the process of spacer selection. We further describe the regulation of adaptation and the protein players involved in this fascinating process that allows bacteria and archaea to harbor adaptive immunity. Copyright © 2016 Elsevier Inc. All rights reserved.

Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9.

Science.gov (United States)

Prykhozhij, Sergey V; Fuller, Charlotte; Steele, Shelby L; Veinotte, Chansey J; Razaghi, Babak; Robitaille, Johane M; McMaster, Christopher R; Shlien, Adam; Malkin, David; Berman, Jason N

2018-06-14

We have optimized point mutation knock-ins into zebrafish genomic sites using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 reagents and single-stranded oligodeoxynucleotides. The efficiency of knock-ins was assessed by a novel application of allele-specific polymerase chain reaction and confirmed by high-throughput sequencing. Anti-sense asymmetric oligo design was found to be the most successful optimization strategy. However, cut site proximity to the mutation and phosphorothioate oligo modifications also greatly improved knock-in efficiency. A previously unrecognized risk of off-target trans knock-ins was identified that we obviated through the development of a workflow for correct knock-in detection. Together these strategies greatly facilitate the study of human genetic diseases in zebrafish, with additional applicability to enhance CRISPR-based approaches in other animal model systems.

FDA Regulation of Clinical Applications of CRISPR-CAS Gene-Editing Technology.

Science.gov (United States)

Grant, Evita V

Scientists have repurposed an adaptive immune system of single cell organisms to create a new type of gene-editing tool: CRISPR (clustered regularly interspaced short palindromic repeats)-Cas technology. Scientists in China have reported its use in the genome modification of non-viable human embryos. This has ignited a spirited debate about the moral, ethical, scientific, and social implications of human germline genome engineering. There have also been calls for regulations; however, FDA has yet to formally announce its oversight of clinical applications of CRISPR-Cas systems. This paper reviews FDA regulation of previously controversial biotechnology breakthroughs, recombinant DNA and human cloning. It then shows that FDA is well positioned to regulate CRISPR-Cas clinical applications, due to its legislative mandates, its existing regulatory frameworks for gene therapies and assisted reproductive technologies, and other considerations.

Palindromic Molecule Beacon-Based Cascade Amplification for Colorimetric Detection of Cancer Genes.

Science.gov (United States)

Shen, Zhi-Fa; Li, Feng; Jiang, Yi-Fan; Chen, Chang; Xu, Huo; Li, Cong-Cong; Yang, Zhe; Wu, Zai-Sheng

2018-03-06

A highly sensitive and selective colorimetric assay based on a multifunctional molecular beacon with palindromic tail (PMB) was proposed for the detection of target p53 gene. The PMB probe can serve as recognition element, primer, and polymerization template and contains a nicking site and a C-rich region complementary to a DNAzyme. In the presence of target DNA, the hairpin of PMB is opened, and the released palindromic tails intermolecularly hybridize with each other, triggering the autonomous polymerization/nicking/displacement cycles. Although only one type of probe is involved, the system can execute triple and continuous polymerization strand displacement amplifications, generating large amounts of G-quadruplex fragments. These G-rich fragments can bind to hemin and form the DNAzymes that possess the catalytic activity similar to horseradish peroxidase, catalyzing the oxidation of ABTS by H 2 O 2 and producing the colorimetric signal. Utilizing the newly proposed sensing system, target DNA can be detected down to 10 pM with a linear response range from 10 pM to 200 nM, and mutant target DNAs are able to be distinguished even by the naked eye. The desirable detection sensitivity, high specificity, and operation convenience without any separation step and chemical modification demonstrate that the palindromic molecular beacon holds the potential for detecting and monitoring a variety of nucleic acid-related biomarkers.

Invasive Species Management on Military Lands: Clustered Regularly Interspaced Short Palindromic Repeat/ CRISPR associated protein 9 (CRISPR/Cas9) based Gene Drives

Science.gov (United States)

2017-06-30

of the crRNA base pairs with tar- get DNA, whereas the 3’ end forms a double- stranded stem with the tra- crRNA to facilitate Cas9 recruitment...or y Ping Gong June 2017 Approved for public release; distribution is unlimited. The U.S. Army Engineer Research and Development...Gong Environmental Laboratory U.S. Army Engineer Research and Development Center 3909 Halls Ferry Road Vicksburg, MS 39180 Final report

Comparative Analysis of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) of Streptococcus thermophilus St-I and its Bacteriophage-Insensitive Mutants (BIM) Derivatives.

Science.gov (United States)

Li, Wan; Bian, Xin; Evivie, Smith Etareri; Huo, Gui-Cheng

2016-09-01

The CRISPR-Cas (CRISPR together with CRISPR-associated proteins) modules are the adaptive immune system, acting as an adaptive and heritable immune system in bacteria and archaea. CRISPR-based immunity acts by integrating short virus sequences in the cell's CRISPR locus, allowing the cell to remember, recognize, and clear infections. In this study, the homology of CRISPRs sequence in BIMs (bacteriophage-insensitive mutants) of Streptococcus thermophilus St-I were analyzed. Secondary structures of the repeats and the PAMs (protospacer-associated motif) of each CRISPR locus were also predicted. Results showed that CRISPR1 has 27 repeat-spacer units, 5 of them had duplicates; CRISPR2 has one repeat-spacer unit; CRISPR3 has 28 repeat-spacer units. Only BIM1 had a new spacer acquisition in CRISPR3, while BIM2 and BIM3 had no new spacers' insertion, thus indicating that while most CRISPR1 were more active than CRISPR3, new spacer acquisition occurred just in CRSPR3 in some situations. These findings will help establish the foundation for the study of CRSPR-Cas systems in lactic acid bacteria.

High hydrostatic pressure adaptive strategies in an obligate piezophile Pyrococcus yayanosii

KAUST Repository

Michoud, Gregoire; Jebbar, Mohamed

2016-01-01

Pyrococcus yayanosii CH1, as the first and only obligate piezophilic hyperthermophilic microorganism discovered to date, extends the physical and chemical limits of life on Earth. It was isolated from the Ashadze hydrothermal vent at 4,100 m depth. Multi-omics analyses were performed to study the mechanisms used by the cell to cope with high hydrostatic pressure variations. In silico analyses showed that the P. yayanosii genome is highly adapted to its harsh environment, with a loss of aromatic amino acid biosynthesis pathways and the high constitutive expression of the energy metabolism compared with other non-obligate piezophilic Pyrococcus species. Differential proteomics and transcriptomics analyses identified key hydrostatic pressure-responsive genes involved in translation, chemotaxis, energy metabolism (hydrogenases and formate metabolism) and Clustered Regularly Interspaced Short Palindromic Repeats sequences associated with Cellular apoptosis susceptibility proteins.

Multiplex conditional mutagenesis in zebrafish using the CRISPR/Cas system.

Science.gov (United States)

Yin, L; Maddison, L A; Chen, W

2016-01-01

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is a powerful tool for genome editing in numerous organisms. However, the system is typically used for gene editing throughout the entire organism. Tissue and temporal specific mutagenesis is often desirable to determine gene function in a specific stage or tissue and to bypass undesired consequences of global mutations. We have developed the CRISPR/Cas system for conditional mutagenesis in transgenic zebrafish using tissue-specific and/or inducible expression of Cas9 and U6-driven expression of sgRNA. To allow mutagenesis of multiple targets, we have isolated four distinct U6 promoters and designed Golden Gate vectors to easily assemble transgenes with multiple sgRNAs. We provide experimental details on the reagents and applications for multiplex conditional mutagenesis in zebrafish. Copyright © 2016 Elsevier Inc. All rights reserved.

High hydrostatic pressure adaptive strategies in an obligate piezophile Pyrococcus yayanosii

KAUST Repository

Michoud, Gregoire

2016-06-02

Pyrococcus yayanosii CH1, as the first and only obligate piezophilic hyperthermophilic microorganism discovered to date, extends the physical and chemical limits of life on Earth. It was isolated from the Ashadze hydrothermal vent at 4,100 m depth. Multi-omics analyses were performed to study the mechanisms used by the cell to cope with high hydrostatic pressure variations. In silico analyses showed that the P. yayanosii genome is highly adapted to its harsh environment, with a loss of aromatic amino acid biosynthesis pathways and the high constitutive expression of the energy metabolism compared with other non-obligate piezophilic Pyrococcus species. Differential proteomics and transcriptomics analyses identified key hydrostatic pressure-responsive genes involved in translation, chemotaxis, energy metabolism (hydrogenases and formate metabolism) and Clustered Regularly Interspaced Short Palindromic Repeats sequences associated with Cellular apoptosis susceptibility proteins.

Mutagenesis and Genome Engineering of Epstein-Barr Virus in Cultured Human Cells by CRISPR/Cas9.

Science.gov (United States)

Yuen, Kit-San; Chan, Chi-Ping; Kok, Kin-Hang; Jin, Dong-Yan

2017-01-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 nuclease (Cas9) system is a powerful genome-editing tool for both chromosomal and extrachromosomal DNA. DNA viruses such as Epstein-Barr virus (EBV), which undergoes episomal replication in human cells, can be effectively edited by CRISPR/Cas9. We have demonstrated targeted editing of the EBV genome by CRISPR/Cas9 in several lines of EBV-infected cells. CRISPR/Cas9-based mutagenesis and genome engineering of EBV provides a new method for genetic analysis, which has some advantages over bacterial artificial chromosome-based recombineering. This approach might also prove useful in the cure of EBV infection. In this chapter, we use the knockout of the BART promoter as an example to detail the experimental procedures for construction of recombinant EBV in human cells.

New technologies accelerate the exploration of non-coding RNAs in horticultural plants

Energy Technology Data Exchange (ETDEWEB)

Liu, Degao; Mewalal, Ritesh; Hu, Rongbin; Tuskan, Gerald A.; Yang, Xiaohan

2017-07-05

Non-coding RNAs (ncRNAs), that is, RNAs not translated into proteins, are crucial regulators of a variety of biological processes in plants. While protein-encoding genes have been relatively well-annotated in sequenced genomes, accounting for a small portion of the genome space in plants, the universe of plant ncRNAs is rapidly expanding. Recent advances in experimental and computational technologies have generated a great momentum for discovery and functional characterization of ncRNAs. Here we summarize the classification and known biological functions of plant ncRNAs, review the application of next-generation sequencing (NGS) technology and ribosome profiling technology to ncRNA discovery in horticultural plants and discuss the application of new technologies, especially the new genome-editing tool clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems, to functional characterization of plant ncRNAs.

Harnessing CRISPR-Cas systems for bacterial genome editing.

Science.gov (United States)

Selle, Kurt; Barrangou, Rodolphe

2015-04-01

Manipulation of genomic sequences facilitates the identification and characterization of key genetic determinants in the investigation of biological processes. Genome editing via clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) constitutes a next-generation method for programmable and high-throughput functional genomics. CRISPR-Cas systems are readily reprogrammed to induce sequence-specific DNA breaks at target loci, resulting in fixed mutations via host-dependent DNA repair mechanisms. Although bacterial genome editing is a relatively unexplored and underrepresented application of CRISPR-Cas systems, recent studies provide valuable insights for the widespread future implementation of this technology. This review summarizes recent progress in bacterial genome editing and identifies fundamental genetic and phenotypic outcomes of CRISPR targeting in bacteria, in the context of tool development, genome homeostasis, and DNA repair. Copyright © 2015 Elsevier Ltd. All rights reserved.

Recent advances in therapeutic recruitment of mammalian RNAi and bacterial CRISPR-Cas DNA interference pathways as emerging antiviral strategies.

Science.gov (United States)

Chin, Wei-Xin; Ang, Swee Kim; Chu, Justin Jang Hann

2017-01-01

In invertebrate eukaryotes and prokaryotes, respectively, the RNAi and clustered regularly interspaced short palindromic repeats-CRISPR-associated (CRISPR-Cas) pathways are highly specific and efficient RNA and DNA interference systems, and are well characterised as potent antiviral systems. It has become possible to recruit or reconstitute these pathways in mammalian cells, where they can be directed against desired host or viral targets. The RNAi and CRISPR-Cas systems can therefore yield ideal antiviral therapeutics, capable of specific and efficient viral inhibition with minimal off-target effects, but development of such therapeutics can be slow. This review covers recent advances made towards developing RNAi or CRISPR-Cas strategies for clinical use. These studies address the delivery, toxicity or target design issues that typically plague the in vivo or clinical use of these technologies. Copyright © 2016 Elsevier Ltd. All rights reserved.

The independent loss model with ordered insertions for the evolution of CRISPR spacers.

Science.gov (United States)

Baumdicker, F; Huebner, A M I; Pfaffelhuber, P

2018-02-01

Today, the CRISPR (clustered regularly interspaced short palindromic repeats) region within bacterial and archaeal genomes is known to encode an adaptive immune system. We rely on previous results on the evolution of the CRISPR arrays, which led to the ordered independent loss model, introduced by Kupczok and Bollback (2013). When focusing on the spacers (between the repeats), new elements enter a CRISPR array at rate θ at the leader end of the array, while all spacers present are lost at rate ρ along the phylogeny relating the sample. Within this model, we compute the distribution of distances of spacers which are present in all arrays in a sample of size n. We use these results to estimate the loss rate ρ from spacer array data for n=2 and n=3. Copyright © 2017 Elsevier Inc. All rights reserved.

CRISPR typing and subtyping for improved laboratory surveillance of Salmonella infections.

Directory of Open Access Journals (Sweden)

Laëtitia Fabre

Full Text Available Laboratory surveillance systems for salmonellosis should ideally be based on the rapid serotyping and subtyping of isolates. However, current typing methods are limited in both speed and precision. Using 783 strains and isolates belonging to 130 serotypes, we show here that a new family of DNA repeats named CRISPR (clustered regularly interspaced short palindromic repeats is highly polymorphic in Salmonella. We found that CRISPR polymorphism was strongly correlated with both serotype and multilocus sequence type. Furthermore, spacer microevolution discriminated between subtypes within prevalent serotypes, making it possible to carry out typing and subtyping in a single step. We developed a high-throughput subtyping assay for the most prevalent serotype, Typhimurium. An open web-accessible database was set up, providing a serotype/spacer dictionary and an international tool for strain tracking based on this innovative, powerful typing and subtyping tool.

Single-step generation of rabbits carrying a targeted allele of the tyrosinase gene using CRISPR/Cas9.

Science.gov (United States)

Honda, Arata; Hirose, Michiko; Sankai, Tadashi; Yasmin, Lubna; Yuzawa, Kazuaki; Honsho, Kimiko; Izu, Haruna; Iguchi, Atsushi; Ikawa, Masahito; Ogura, Atsuo

2015-01-01

Targeted genome editing of nonrodent mammalian species has provided the potential for highly accurate interventions into gene function in humans and the generation of useful animal models of human diseases. Here we show successful clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas)-mediated gene targeting via circular plasmid injection in rabbits. The rabbit tyrosinase gene (TYR) was effectively disrupted, and we confirmed germline transmission by pronuclear injection of a circular plasmid expressing humanized Cas9 (hCas9) and single-guide RNA. Direct injection into pronuclear stage zygotes was possible following an in vitro validation assay. Neither off-target mutagenesis nor hCas9 transgenesis was detected in any of the genetically targeted pups and embryos examined. Gene targeting with this rapid and simplified strategy will help accelerate the development of translational research using other nonrodent mammalian species.

CRISPR-Cas: From the Bacterial Adaptive Immune System to a Versatile Tool for Genome Engineering.

Science.gov (United States)

Kirchner, Marion; Schneider, Sabine

2015-11-09

The field of biology has been revolutionized by the recent advancement of an adaptive bacterial immune system as a universal genome engineering tool. Bacteria and archaea use repetitive genomic elements termed clustered regularly interspaced short palindromic repeats (CRISPR) in combination with an RNA-guided nuclease (CRISPR-associated nuclease: Cas) to target and destroy invading DNA. By choosing the appropriate sequence of the guide RNA, this two-component system can be used to efficiently modify, target, and edit genomic loci of interest in plants, insects, fungi, mammalian cells, and whole organisms. This has opened up new frontiers in genome engineering, including the potential to treat or cure human genetic disorders. Now the potential risks as well as the ethical, social, and legal implications of this powerful new technique move into the limelight. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria.

Science.gov (United States)

Yosef, Ido; Manor, Miriam; Kiro, Ruth; Qimron, Udi

2015-06-09

The increasing threat of pathogen resistance to antibiotics requires the development of novel antimicrobial strategies. Here we present a proof of concept for a genetic strategy that aims to sensitize bacteria to antibiotics and selectively kill antibiotic-resistant bacteria. We use temperate phages to deliver a functional clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system into the genome of antibiotic-resistant bacteria. The delivered CRISPR-Cas system destroys both antibiotic resistance-conferring plasmids and genetically modified lytic phages. This linkage between antibiotic sensitization and protection from lytic phages is a key feature of the strategy. It allows programming of lytic phages to kill only antibiotic-resistant bacteria while protecting antibiotic-sensitized bacteria. Phages designed according to this strategy may be used on hospital surfaces and hand sanitizers to facilitate replacement of antibiotic-resistant pathogens with sensitive ones.

Harnessing type I and type III CRISPR-Cas systems for genome editing

DEFF Research Database (Denmark)

Li, Yingjun; Pan, Saifu; Zhang, Yan

2016-01-01

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are widespread in archaea and bacteria, and research on their molecular mechanisms has led to the development of genome-editing techniques based on a few Type II systems. However, there has not been any...... report on harnessing a Type I or Type III system for genome editing. Here, a method was developed to repurpose both CRISPR-Cas systems for genetic manipulation in Sulfolobus islandicus, a thermophilic archaeon. A novel type of genome-editing plasmid (pGE) was constructed, carrying an artificial mini-CRISPR...... and selectively retained as transformants. Using this strategy, different types of mutation were generated, including deletion, insertion and point mutations. We envision this method is readily applicable to different bacteria and archaea that carry an active CRISPR-Cas system of DNA interference provided...

Generation of an ICF Syndrome Model by Efficient Genome Editing of Human Induced Pluripotent Stem Cells Using the CRISPR System

Directory of Open Access Journals (Sweden)

Izuho Hatada

2013-09-01

Full Text Available Genome manipulation of human induced pluripotent stem (iPS cells is essential to achieve their full potential as tools for regenerative medicine. To date, however, gene targeting in human pluripotent stem cells (hPSCs has proven to be extremely difficult. Recently, an efficient genome manipulation technology using the RNA-guided DNase Cas9, the clustered regularly interspaced short palindromic repeats (CRISPR system, has been developed. Here we report the efficient generation of an iPS cell model for immunodeficiency, centromeric region instability, facial anomalies syndrome (ICF syndrome using the CRISPR system. We obtained iPS cells with mutations in both alleles of DNA methyltransferase 3B (DNMT3B in 63% of transfected clones. Our data suggest that the CRISPR system is highly efficient and useful for genome engineering of human iPS cells.

Genome editing: a robust technology for human stem cells.

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Chandrasekaran, Arun Pandian; Song, Minjung; Ramakrishna, Suresh

2017-09-01

Human pluripotent stem cells comprise induced pluripotent and embryonic stem cells, which have tremendous potential for biological and therapeutic applications. The development of efficient technologies for the targeted genome alteration of stem cells in disease models is a prerequisite for utilizing stem cells to their full potential. Genome editing of stem cells is possible with the help of synthetic nucleases that facilitate site-specific modification of a gene of interest. Recent advances in genome editing techniques have improved the efficiency and speed of the development of stem cells for human disease models. Zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system are powerful tools for editing DNA at specific loci. Here, we discuss recent technological advances in genome editing with site-specific nucleases in human stem cells.

Site-Specific Integration of Exogenous Genes Using Genome Editing Technologies in Zebrafish

Directory of Open Access Journals (Sweden)

Atsuo Kawahara

2016-05-01

Full Text Available The zebrafish (Danio rerio is an ideal vertebrate model to investigate the developmental molecular mechanism of organogenesis and regeneration. Recent innovation in genome editing technologies, such as zinc finger nucleases (ZFNs, transcription activator-like effector nucleases (TALENs and the clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR associated protein 9 (Cas9 system, have allowed researchers to generate diverse genomic modifications in whole animals and in cultured cells. The CRISPR/Cas9 and TALEN techniques frequently induce DNA double-strand breaks (DSBs at the targeted gene, resulting in frameshift-mediated gene disruption. As a useful application of genome editing technology, several groups have recently reported efficient site-specific integration of exogenous genes into targeted genomic loci. In this review, we provide an overview of TALEN- and CRISPR/Cas9-mediated site-specific integration of exogenous genes in zebrafish.

High hydrostatic pressure adaptive strategies in an obligate piezophile Pyrococcus yayanosii

Science.gov (United States)

Michoud, Grégoire; Jebbar, Mohamed

2016-01-01

Pyrococcus yayanosii CH1, as the first and only obligate piezophilic hyperthermophilic microorganism discovered to date, extends the physical and chemical limits of life on Earth. It was isolated from the Ashadze hydrothermal vent at 4,100 m depth. Multi-omics analyses were performed to study the mechanisms used by the cell to cope with high hydrostatic pressure variations. In silico analyses showed that the P. yayanosii genome is highly adapted to its harsh environment, with a loss of aromatic amino acid biosynthesis pathways and the high constitutive expression of the energy metabolism compared with other non-obligate piezophilic Pyrococcus species. Differential proteomics and transcriptomics analyses identified key hydrostatic pressure-responsive genes involved in translation, chemotaxis, energy metabolism (hydrogenases and formate metabolism) and Clustered Regularly Interspaced Short Palindromic Repeats sequences associated with Cellular apoptosis susceptibility proteins. PMID:27250364

Taming Parasites by Tailoring Them

Directory of Open Access Journals (Sweden)

Bingjian Ren

2017-07-01

Full Text Available The next-generation gene editing based on CRISPR (clustered regularly interspaced short palindromic repeats has been successfully implemented in a wide range of organisms including some protozoan parasites. However, application of such a versatile game-changing technology in molecular parasitology remains fairly underexplored. Here, we briefly introduce state-of-the-art in human and mouse research and usher new directions to drive the parasitology research in the years to come. In precise, we outline contemporary ways to embolden existing apicomplexan and kinetoplastid parasite models by commissioning front-line gene-tailoring methods, and illustrate how we can break the enduring gridlock of gene manipulation in non-model parasitic protists to tackle intriguing questions that remain long unresolved otherwise. We show how a judicious solicitation of the CRISPR technology can eventually balance out the two facets of pathogen-host interplay.

MATLAB Algorithms for Rapid Detection and Embedding of Palindrome and Emordnilap Electronic Watermarks in Simulated Chemical and Biological Image Data

National Research Council Canada - National Science Library

Robbins, Ronny C

2004-01-01

.... This is similar to words such as STOP which when flipped left right gives the new word POTS. Emordnilap is palindrome spelled backwards. This paper explores the use of MATLAB algorithms in the rapid detection and embedding of palindrome and emordnilap electronic watermarks in simulated chemical and biological Image Data.

Single-Molecule View of Small RNA-Guided Target Search and Recognition.

Science.gov (United States)

Globyte, Viktorija; Kim, Sung Hyun; Joo, Chirlmin

2018-05-20

Most everyday processes in life involve a necessity for an entity to locate its target. On a cellular level, many proteins have to find their target to perform their function. From gene-expression regulation to DNA repair to host defense, numerous nucleic acid-interacting proteins use distinct target search mechanisms. Several proteins achieve that with the help of short RNA strands known as guides. This review focuses on single-molecule advances studying the target search and recognition mechanism of Argonaute and CRISPR (clustered regularly interspaced short palindromic repeats) systems. We discuss different steps involved in search and recognition, from the initial complex prearrangement into the target-search competent state to the final proofreading steps. We focus on target search mechanisms that range from weak interactions, to one- and three-dimensional diffusion, to conformational proofreading. We compare the mechanisms of Argonaute and CRISPR with a well-studied target search system, RecA.

TALEN- and CRISPR/Cas9-Mediated Gene Editing in Human Pluripotent Stem Cells Using Lipid-Based Transfection.

Science.gov (United States)

Hendriks, William T; Jiang, Xin; Daheron, Laurence; Cowan, Chad A

2015-08-03

Using custom-engineered nuclease-mediated genome editing, such as Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) RNA-guided Cas9 nucleases, human pluripotent stem cell (hPSC) lines with knockout or mutant alleles can be generated and differentiated into various cell types. This strategy of genome engineering in hPSCs will prove invaluable for studying human biology and disease. Here, we provide a detailed protocol for design and construction of TALEN and CRISPR vectors, testing of their nuclease activity, and delivery of TALEN or CRISPR vectors into hPSCs. In addition, we describe the use of single-stranded oligodeoxynucleotides (ssODNs) to introduce or repair point mutations. Next, we describe the identification of edited hPSC clones without antibiotic selection, including their clonal selection, genotyping, and expansion for downstream applications. Copyright © 2015 John Wiley & Sons, Inc.

The potential of the combination of CRISPR/Cas9 and pluripotent stem cells to provide human organs from chimaeric pigs.

Science.gov (United States)

Feng, Wanyou; Dai, Yifan; Mou, Lisha; Cooper, David K C; Shi, Deshun; Cai, Zhiming

2015-03-23

Clinical organ allotransplantation is limited by the availability of deceased human donors. However, the transplantation of human organs produced in other species would provide an unlimited number of organs. The pig has been identified as the most suitable source of organs for humans as organs of any size would be available. Genome editing by RNA-guided endonucleases, also known as clustered regularly interspaced short palindromic repeat (CRISPR/Cas9), in combination with induced pluripotent stem cells (iPSC), may have the potential to enable the creation of human organs from genetically-modified chimaeric pigs. These could potentially provide an unlimited supply of organs that would not be rejected by the recipient's immune system. However, substantial research is needed to prove that this approach will work. Genetic modification of chimaeric pigs could also provide useful models for developing therapies for various human diseases, especially in relation to drug development.

A CRISPR CASe for High-Throughput Silencing

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Jacob eHeintze

2013-10-01

Full Text Available Manipulation of gene expression on a genome-wide level is one of the most important systematic tools in the post-genome era. Such manipulations have largely been enabled by expression cloning approaches using sequence-verified cDNA libraries, large-scale RNA interference libraries (shRNA or siRNA and zinc finger nuclease technologies. More recently, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated (Cas9-mediated gene editing technology has been described that holds great promise for future use of this technology in genomic manipulation. It was suggested that the CRISPR system has the potential to be used in high-throughput, large-scale loss of function screening. Here we discuss some of the challenges in engineering of CRISPR/Cas genomic libraries and some of the aspects that need to be addressed in order to use this technology on a high-throughput scale.

Applications of Alternative Nucleases in the Age of CRISPR/Cas9

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Tuhin K. Guha

2017-11-01

Full Text Available Breakthroughs in the development of programmable site-specific nucleases, including zinc-finger nucleases (ZFNs, transcription activator-like effector nucleases (TALENs, meganucleases (MNs, and most recently, the clustered regularly interspaced short palindromic repeats (CRISPR associated proteins (including Cas9 have greatly enabled and accelerated genome editing. By targeting double-strand breaks to user-defined locations, the rates of DNA repair events are greatly enhanced relative to un-catalyzed events at the same sites. However, the underlying biology of each genome-editing nuclease influences the targeting potential, the spectrum of off-target cleavages, the ease-of-use, and the types of recombination events at targeted double-strand breaks. No single genome-editing nuclease is optimized for all possible applications. Here, we focus on the diversity of nuclease domains available for genome editing, highlighting biochemical properties and the potential applications that are best suited to each domain.

Genetic Studies on CRISPR-Cas Functions in Invader Defense in Sulfolobus islandicus

DEFF Research Database (Denmark)

Peng, Wenfang

Archaea and bacteria contain CRISPR-Cas (clustered regularly interspaced short palindromic repeat-CRISPR-associated) systems that protect themselves against invasion by viruses and plasmids. There are three major types of CRISPR-Cas systems, type I, II and III, that are further divided...... into at least 11 subtypes. I employed Sulfolobus islandicus Rey15A as the model to study CRISPR mechanisms. The model archaeon encodes one subtype I-A (Cascade) and two subtype III-B (Cmr-α and Cmr-β) interference systems with no apparent redundancy in cas genes or in CRISPR systems, which is ideal for genetic...... analysis of cas gene function. Furthermore, a range of genetic tools have been developed for S. islandicus Rey15A in our laboratory and a plasmid interference assay has been successfully developed for testing CRISPR-directed DNA targeting activity, which have provided a solid basis for studying...

Highly efficient biallelic genome editing of human ES/iPS cells using a CRISPR/Cas9 or TALEN system.

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Takayama, Kazuo; Igai, Keisuke; Hagihara, Yasuko; Hashimoto, Rina; Hanawa, Morifumi; Sakuma, Tetsushi; Tachibana, Masashi; Sakurai, Fuminori; Yamamoto, Takashi; Mizuguchi, Hiroyuki

2017-05-19

Genome editing research of human ES/iPS cells has been accelerated by clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) and transcription activator-like effector nucleases (TALEN) technologies. However, the efficiency of biallelic genetic engineering in transcriptionally inactive genes is still low, unlike that in transcriptionally active genes. To enhance the biallelic homologous recombination efficiency in human ES/iPS cells, we performed screenings of accessorial genes and compounds. We found that RAD51 overexpression and valproic acid treatment enhanced biallelic-targeting efficiency in human ES/iPS cells regardless of the transcriptional activity of the targeted locus. Importantly, RAD51 overexpression and valproic acid treatment synergistically increased the biallelic homologous recombination efficiency. Our findings would facilitate genome editing study using human ES/iPS cells. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

A Genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Screen Identifies NEK7 as an Essential Component of NLRP3 Inflammasome Activation.

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Schmid-Burgk, Jonathan L; Chauhan, Dhruv; Schmidt, Tobias; Ebert, Thomas S; Reinhardt, Julia; Endl, Elmar; Hornung, Veit

2016-01-01

Inflammasomes are high molecular weight protein complexes that assemble in the cytosol upon pathogen encounter. This results in caspase-1-dependent pro-inflammatory cytokine maturation, as well as a special type of cell death, known as pyroptosis. The Nlrp3 inflammasome plays a pivotal role in pathogen defense, but at the same time, its activity has also been implicated in many common sterile inflammatory conditions. To this effect, several studies have identified Nlrp3 inflammasome engagement in a number of common human diseases such as atherosclerosis, type 2 diabetes, Alzheimer disease, or gout. Although it has been shown that known Nlrp3 stimuli converge on potassium ion efflux upstream of Nlrp3 activation, the exact molecular mechanism of Nlrp3 activation remains elusive. Here, we describe a genome-wide CRISPR/Cas9 screen in immortalized mouse macrophages aiming at the unbiased identification of gene products involved in Nlrp3 inflammasome activation. We employed a FACS-based screen for Nlrp3-dependent cell death, using the ionophoric compound nigericin as a potassium efflux-inducing stimulus. Using a genome-wide guide RNA (gRNA) library, we found that targeting Nek7 rescued macrophages from nigericin-induced lethality. Subsequent studies revealed that murine macrophages deficient in Nek7 displayed a largely blunted Nlrp3 inflammasome response, whereas Aim2-mediated inflammasome activation proved to be fully intact. Although the mechanism of Nek7 functioning upstream of Nlrp3 yet remains elusive, these studies provide a first genetic handle of a component that specifically functions upstream of Nlrp3. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Palindromic nucleotide analysis in human T cell receptor rearrangements.

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Santosh K Srivastava

Full Text Available Diversity of T cell receptor (TCR genes is primarily generated by nucleotide insertions upon rearrangement from their germ line-encoded V, D and J segments. Nucleotide insertions at V-D and D-J junctions are random, but some small subsets of these insertions are exceptional, in that one to three base pairs inversely repeat the sequence of the germline DNA. These short complementary palindromic sequences are called P nucleotides. We apply the ImmunoSeq deep-sequencing assay to the third complementarity determining region (CDR3 of the β chain of T cell receptors, and use the resulting data to study P nucleotides in the repertoire of naïve and memory CD8(+ and CD4(+ T cells. We estimate P nucleotide distributions in a cross section of healthy adults and different T cell subtypes. We show that P nucleotide frequency in all T cell subtypes ranges from 1% to 2%, and that the distribution is highly biased with respect to the coding end of the gene segment. Classification of observed palindromic sequences into P nucleotides using a maximum conditional probability model shows that single base P nucleotides are very rare in VDJ recombination; P nucleotides are primarily two bases long. To explore the role of P nucleotides in thymic selection, we compare P nucleotides in productive and non-productive sequences of CD8(+ naïve T cells. The naïve CD8(+ T cell clones with P nucleotides are more highly expanded.

Trapping of palindromic ligands within native transthyretin prevents amyloid formation

OpenAIRE

Kolstoe, Simon E.; Mangione, Palma P.; Bellotti, Vittorio; Taylor, Graham W.; Tennent, Glenys A.; Deroo, Stéphanie; Morrison, Angus J.; Cobb, Alexander J. A.; Coyne, Anthony; McCammon, Margaret G.; Warner, Timothy D.; Mitchell, Jane; Gill, Raj; Smith, Martin D.; Ley, Steven V.

2010-01-01

Transthyretin (TTR) amyloidosis is a fatal disease for which new therapeutic approaches are urgently needed. We have designed two palindromic ligands, 2,2'-(4,4'-(heptane-1,7-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (mds84) and 2,2'-(4,4'-(undecane-1,11-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (4ajm15), that are rapidly bound by native wild-type TTR in whole serum and even more avidly by amyloidogenic TTR variants. One to one...

International regulatory landscape and integration of corrective genome editing into in vitro fertilization.

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Araki, Motoko; Ishii, Tetsuya

2014-11-24

Genome editing technology, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas, has enabled far more efficient genetic engineering even in non-human primates. This biotechnology is more likely to develop into medicine for preventing a genetic disease if corrective genome editing is integrated into assisted reproductive technology, represented by in vitro fertilization. Although rapid advances in genome editing are expected to make germline gene correction feasible in a clinical setting, there are many issues that still need to be addressed before this could occur. We herein examine current status of genome editing in mammalian embryonic stem cells and zygotes and discuss potential issues in the international regulatory landscape regarding human germline gene modification. Moreover, we address some ethical and social issues that would be raised when each country considers whether genome editing-mediated germline gene correction for preventive medicine should be permitted.

Multimode drug inducible CRISPR/Cas9 devices for transcriptional activation and genome editing

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Lu, Jia; Zhao, Chen; Zhao, Yingze; Zhang, Jingfang; Zhang, Yue; Chen, Li; Han, Qiyuan; Ying, Yue; Peng, Shuai; Ai, Runna; Wang, Yu

2018-01-01

Abstract Precise investigation and manipulation of dynamic biological processes often requires molecular modulation in a controlled inducible manner. The clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) has emerged as a versatile tool for targeted gene editing and transcriptional programming. Here, we designed and vigorously optimized a series of Hybrid drug Inducible CRISPR/Cas9 Technologies (HIT) for transcriptional activation by grafting a mutated human estrogen receptor (ERT2) to multiple CRISPR/Cas9 systems, which renders them 4-hydroxytamoxifen (4-OHT) inducible for the access of genome. Further, extra functionality of simultaneous genome editing was achieved with one device we named HIT2. Optimized terminal devices herein delivered advantageous performances in comparison with several existing designs. They exerted selective, titratable, rapid and reversible response to drug induction. In addition, these designs were successfully adapted to an orthogonal Cas9. HIT systems developed in this study can be applied for controlled modulation of potentially any genomic loci in multiple modes. PMID:29237052

CRISPR and the Rebirth of Synthetic Biology.

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Heidari, Raheleh; Shaw, David Martin; Elger, Bernice Simone

2017-04-01

Emergence of novel genome engineering technologies such as clustered regularly interspaced short palindromic repeat (CRISPR) has refocused attention on unresolved ethical complications of synthetic biology. Biosecurity concerns, deontological issues and human right aspects of genome editing have been the subject of in-depth debate; however, a lack of transparent regulatory guidelines, outdated governance codes, inefficient time-consuming clinical trial pathways and frequent misunderstanding of the scientific potential of cutting-edge technologies have created substantial obstacles to translational research in this area. While a precautionary principle should be applied at all stages of genome engineering research, the stigma of germline editing, synthesis of new life forms and unrealistic presentation of current technologies should not arrest the transition of new therapeutic, diagnostic or preventive tools from research to clinic. We provide a brief review on the present regulation of CRISPR and discuss the translational aspect of genome engineering research and patient autonomy with respect to the "right to try" potential novel non-germline gene therapies.

Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells.

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Liao, Hsin-Kai; Gu, Ying; Diaz, Arturo; Marlett, John; Takahashi, Yuta; Li, Mo; Suzuki, Keiichiro; Xu, Ruo; Hishida, Tomoaki; Chang, Chan-Jung; Esteban, Concepcion Rodriguez; Young, John; Izpisua Belmonte, Juan Carlos

2015-03-10

To combat hostile viruses, bacteria and archaea have evolved a unique antiviral defense system composed of clustered regularly interspaced short palindromic repeats (CRISPRs), together with CRISPR-associated genes (Cas). The CRISPR/Cas9 system develops an adaptive immune resistance to foreign plasmids and viruses by creating site-specific DNA double-stranded breaks (DSBs). Here we adapt the CRISPR/Cas9 system to human cells for intracellular defense against foreign DNA and viruses. Using HIV-1 infection as a model, our results demonstrate that the CRISPR/Cas9 system disrupts latently integrated viral genome and provides long-term adaptive defense against new viral infection, expression and replication in human cells. We show that engineered human-induced pluripotent stem cells stably expressing HIV-targeted CRISPR/Cas9 can be efficiently differentiated into HIV reservoir cell types and maintain their resistance to HIV-1 challenge. These results unveil the potential of the CRISPR/Cas9 system as a new therapeutic strategy against viral infections.

Induced mutation and epigenetics modification in plants for crop improvement by targeting CRISPR/Cas9 technology.

Science.gov (United States)

Khan, Muhammad Hafeez Ullah; Khan, Shahid U; Muhammad, Ali; Hu, Limin; Yang, Yang; Fan, Chuchuan

2018-06-01

Clustered regularly interspaced palindromic repeats associated protein Cas9 (CRISPR-Cas9), originally an adaptive immunity system of prokaryotes, is revolutionizing genome editing technologies with minimal off-targets in the present era. The CRISPR/Cas9 is now highly emergent, advanced, and highly specific tool for genome engineering. The technology is widely used to animal and plant genomes to achieve desirable results. The present review will encompass how CRISPR-Cas9 is revealing its beneficial role in characterizing plant genetic functions, genomic rearrangement, how it advances the site-specific mutagenesis, and epigenetics modification in plants to improve the yield of field crops with minimal side-effects. The possible pitfalls of using and designing CRISPR-Cas9 for plant genome editing are also discussed for its more appropriate applications in plant biology. Therefore, CRISPR/Cas9 system has multiple benefits that mostly scientists select for genome editing in several biological systems. © 2017 Wiley Periodicals, Inc.

CRISPR/Cas9 Platforms for Genome Editing in Plants: Developments and Applications.

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Ma, Xingliang; Zhu, Qinlong; Chen, Yuanling; Liu, Yao-Guang

2016-07-06

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein9 (Cas9) genome editing system (CRISPR/Cas9) is adapted from the prokaryotic type II adaptive immunity system. The CRISPR/Cas9 tool surpasses other programmable nucleases, such as ZFNs and TALENs, for its simplicity and high efficiency. Various plant-specific CRISPR/Cas9 vector systems have been established for adaption of this technology to many plant species. In this review, we present an overview of current advances on applications of this technology in plants, emphasizing general considerations for establishment of CRISPR/Cas9 vector platforms, strategies for multiplex editing, methods for analyzing the induced mutations, factors affecting editing efficiency and specificity, and features of the induced mutations and applications of the CRISPR/Cas9 system in plants. In addition, we provide a perspective on the challenges of CRISPR/Cas9 technology and its significance for basic plant research and crop genetic improvement. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

Synthetic RNAs for Gene Regulation: Design Principles and Computational Tools

International Nuclear Information System (INIS)

Laganà, Alessandro; Shasha, Dennis; Croce, Carlo Maria

2014-01-01

The use of synthetic non-coding RNAs for post-transcriptional regulation of gene expression has not only become a standard laboratory tool for gene functional studies but it has also opened up new perspectives in the design of new and potentially promising therapeutic strategies. Bioinformatics has provided researchers with a variety of tools for the design, the analysis, and the evaluation of RNAi agents such as small-interfering RNA (siRNA), short-hairpin RNA (shRNA), artificial microRNA (a-miR), and microRNA sponges. More recently, a new system for genome engineering based on the bacterial CRISPR-Cas9 system (Clustered Regularly Interspaced Short Palindromic Repeats), was shown to have the potential to also regulate gene expression at both transcriptional and post-transcriptional level in a more specific way. In this mini review, we present RNAi and CRISPRi design principles and discuss the advantages and limitations of the current design approaches.

CRISPR-Cas9 technology and its application in haematological disorders

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Zhang, Han; McCarty, Nami

2018-01-01

Summary The recent advent of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein 9 (Cas9) system for precise genome editing has revolutionized methodologies in haematology and oncology studies. CRISPR-Cas9 technology can be used to remove and correct genes or mutations, and to introduce site-specific therapeutic genes in human cells. Inherited haematological disorders represent ideal targets for CRISPR-Cas9-mediated gene therapy. Correcting disease-causing mutations could alleviate disease-related symptoms in the near future. The CRISPR-Cas9 system is also a useful tool for delineating molecular mechanisms involving haematological malignancies. Prior to the use of CRISPR-Cas9-mediated gene correction in humans, appropriate delivery systems with higher efficiency and specificity must be identified, and ethical guidelines for applying the technology with controllable safety must be established. Here, the latest applications of CRISPR-Cas9 technology in haematological disorders, current challenges and future directions are reviewed and discussed. PMID:27619566

Genome editing: the road of CRISPR/Cas9 from bench to clinic

KAUST Repository

Eid, Ayman

2016-10-14

Molecular scissors engineered for site-specific modification of the genome hold great promise for effective functional analyses of genes, genomes and epigenomes and could improve our understanding of the molecular underpinnings of disease states and facilitate novel therapeutic applications. Several platforms for molecular scissors that enable targeted genome engineering have been developed, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and, most recently, clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated-9 (Cas9). The CRISPR/Cas9 system\\'s simplicity, facile engineering and amenability to multiplexing make it the system of choice for many applications. CRISPR/Cas9 has been used to generate disease models to study genetic diseases. Improvements are urgently needed for various aspects of the CRISPR/Cas9 system, including the system\\'s precision, delivery and control over the outcome of the repair process. Here, we discuss the current status of genome engineering and its implications for the future of biological research and gene therapy.

Mathematical modelling of CRISPR-Cas system effects on biofilm formation.

Science.gov (United States)

Ali, Qasim; Wahl, Lindi M

2017-08-01

Clustered regularly interspaced short palindromic repeats (CRISPR), linked with CRISPR associated (Cas) genes, can confer adaptive immunity to bacteria, against bacteriophage infections. Thus from a therapeutic standpoint, CRISPR immunity increases biofilm resistance to phage therapy. Recently, however, CRISPR-Cas genes have been implicated in reducing biofilm formation in lysogenized cells. Thus CRISPR immunity can have complex effects on phage-host-lysogen interactions, particularly in a biofilm. In this contribution, we develop and analyse a series of dynamical systems to elucidate and disentangle these interactions. Two competition models are used to study the effects of lysogens (first model) and CRISPR-immune bacteria (second model) in the biofilm. In the third model, the effect of delivering lysogens to a CRISPR-immune biofilm is investigated. Using standard analyses of equilibria, stability and bifurcations, our models predict that lysogens may be able to displace CRISPR-immune bacteria in a biofilm, and thus suggest strategies to eliminate phage-resistant biofilms.

CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity.

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Barrangou, Rodolphe; Marraffini, Luciano A

2014-04-24

Clustered regularly interspaced short palindromic repeats (CRISPR), and associated proteins (Cas) comprise the CRISPR-Cas system, which confers adaptive immunity against exogenic elements in many bacteria and most archaea. CRISPR-mediated immunization occurs through the uptake of DNA from invasive genetic elements such as plasmids and viruses, followed by its integration into CRISPR loci. These loci are subsequently transcribed and processed into small interfering RNAs that guide nucleases for specific cleavage of complementary sequences. Conceptually, CRISPR-Cas shares functional features with the mammalian adaptive immune system, while also exhibiting characteristics of Lamarckian evolution. Because immune markers spliced from exogenous agents are integrated iteratively in CRISPR loci, they constitute a genetic record of vaccination events and reflect environmental conditions and changes over time. Cas endonucleases, which can be reprogrammed by small guide RNAs have shown unprecedented potential and flexibility for genome editing and can be repurposed for numerous DNA targeting applications including transcriptional control. Copyright © 2014 Elsevier Inc. All rights reserved.

Target motifs affecting natural immunity by a constitutive CRISPR-Cas system in Escherichia coli.

Directory of Open Access Journals (Sweden)

Cristóbal Almendros

Full Text Available Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR and CRISPR associated (cas genes conform the CRISPR-Cas systems of various bacteria and archaea and produce degradation of invading nucleic acids containing sequences (protospacers that are complementary to repeat intervening spacers. It has been demonstrated that the base sequence identity of a protospacer with the cognate spacer and the presence of a protospacer adjacent motif (PAM influence CRISPR-mediated interference efficiency. By using an original transformation assay with plasmids targeted by a resident spacer here we show that natural CRISPR-mediated immunity against invading DNA occurs in wild type Escherichia coli. Unexpectedly, the strongest activity is observed with protospacer adjoining nucleotides (interference motifs that differ from the PAM both in sequence and location. Hence, our results document for the first time native CRISPR activity in E. coli and demonstrate that positions next to the PAM in invading DNA influence their recognition and degradation by these prokaryotic immune systems.

Evolution and classification of the CRISPR-Cas systems

Science.gov (United States)

S. Makarova, Kira; H. Haft, Daniel; Barrangou, Rodolphe; J. J. Brouns, Stan; Charpentier, Emmanuelle; Horvath, Philippe; Moineau, Sylvain; J. M. Mojica, Francisco; I. Wolf, Yuri; Yakunin, Alexander F.; van der Oost, John; V. Koonin, Eugene

2012-01-01

The CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) modules are adaptive immunity systems that are present in many archaea and bacteria. These defence systems are encoded by operons that have an extraordinarily diverse architecture and a high rate of evolution for both the cas genes and the unique spacer content. Here, we provide an updated analysis of the evolutionary relationships between CRISPR–Cas systems and Cas proteins. Three major types of CRISPR–Cas system are delineated, with a further division into several subtypes and a few chimeric variants. Given the complexity of the genomic architectures and the extremely dynamic evolution of the CRISPR–Cas systems, a unified classification of these systems should be based on multiple criteria. Accordingly, we propose a `polythetic' classification that integrates the phylogenies of the most common cas genes, the sequence and organization of the CRISPR repeats and the architecture of the CRISPR–cas loci. PMID:21552286

Simple Genome Editing of Rodent Intact Embryos by Electroporation.

Directory of Open Access Journals (Sweden)

Takehito Kaneko

Full Text Available The clustered regularly interspaced short palindromic repeat (CRISPR/CRISPR-associated (Cas system is a powerful tool for genome editing in animals. Recently, new technology has been developed to genetically modify animals without using highly skilled techniques, such as pronuclear microinjection of endonucleases. Technique for animal knockout system by electroporation (TAKE method is a simple and effective technology that produces knockout rats by introducing endonuclease mRNAs into intact embryos using electroporation. Using TAKE method and CRISPR/Cas system, the present study successfully produced knockout and knock-in mice and rats. The mice and rats derived from embryos electroporated with Cas9 mRNA, gRNA and single-stranded oligodeoxynucleotide (ssODN comprised the edited targeted gene as a knockout (67% of mice and 88% of rats or knock-in (both 33%. The TAKE method could be widely used as a powerful tool to produce genetically modified animals by genome editing.

An enrichment of CRISPR and other defense-related features in marine sponge-associated microbial metagenomes

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Hannes Horn

2016-11-01

Full Text Available Many marine sponges are populated by dense and taxonomically diverse microbial consortia. We employed a metagenomics approach to unravel the differences in the functional gene repertoire among three Mediterranean sponge species, Petrosia ficiformis, Sarcotragus foetidus, Aplysina aerophoba and seawater. Different signatures were observed between sponge and seawater metagenomes with regard to microbial community composition, GC content, and estimated bacterial genome size. Our analysis showed further a pronounced repertoire for defense systems in sponge metagenomes. Specifically, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR, restriction modification, DNA phosphorothioation and phage growth limitation systems were enriched in sponge metagenomes. These data suggest that defense is an important functional trait for an existence within sponges that requires mechanisms to defend against foreign DNA from microorganisms and viruses. This study contributes to an understanding of the evolutionary arms race between viruses/phages and bacterial genomes and it sheds light on the bacterial defenses that have evolved in the context of the sponge holobiont.

Whole genome sequence analysis of Geitlerinema sp. FC II unveils competitive edge of the strain in marine cultivation system for biofuel production.

Science.gov (United States)

Batchu, Navish Kumar; Khater, Shradha; Patil, Sonal; Nagle, Vinod; Das, Gautam; Bhadra, Bhaskar; Sapre, Ajit; Dasgupta, Santanu

2018-03-05

A filamentous cyanobacteria, Geitlerinema sp. FC II, was isolated from marine algae culture pond at Reliance Industries Limited (RIL), India. The 6.7 Mb draft genome of FC II encodes for 6697 protein coding genes. Analysis of the whole genome sequence revealed presence of nif gene cluster, supporting its capability to fix atmospheric nitrogen. FC II genome contains two variants of sulfide:quinone oxidoreductases (SQR), which is a crucial elector donor in cyanobacterial metabolic processes. FC II is characterized by the presence of multiple CRISPR- Cas (Clustered Regularly Interspaced Short Palindrome Repeats - CRISPR associated proteins) clusters, multiple variants of genes encoding photosystem reaction centres, biosynthetic gene clusters of alkane, polyketides and non-ribosomal peptides. Presence of these pathways will help FC II in gaining an ecological advantage over other strains for biomass production in large scale cultivation system. Hence, FC II may be used for production of biofuel and other industrially important metabolites. Copyright © 2018 Elsevier Inc. All rights reserved.

Engineering Molecular Immunity Against Plant Viruses

KAUST Repository

Zaidi, Syed Shan-e-Ali; Tashkandi, Manal; Mahfouz, Magdy M.

2017-01-01

Genomic engineering has been used to precisely alter eukaryotic genomes at the single-base level for targeted gene editing, replacement, fusion, and mutagenesis, and plant viruses such as Tobacco rattle virus have been developed into efficient vectors for delivering genome-engineering reagents. In addition to altering the host genome, these methods can target pathogens to engineer molecular immunity. Indeed, recent studies have shown that clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) systems that target the genomes of DNA viruses can interfere with viral activity and limit viral symptoms in planta, demonstrating the utility of this system for engineering molecular immunity in plants. CRISPR/Cas9 can efficiently target single and multiple viral infections and confer plant immunity. Here, we discuss the use of site-specific nucleases to engineer molecular immunity against DNA and RNA viruses in plants. We also explore how to address the potential challenges encountered when producing plants with engineered resistance to single and mixed viral infections.

Engineering Molecular Immunity Against Plant Viruses

KAUST Repository

Zaidi, Syed Shan-e-Ali

2017-04-26

Genomic engineering has been used to precisely alter eukaryotic genomes at the single-base level for targeted gene editing, replacement, fusion, and mutagenesis, and plant viruses such as Tobacco rattle virus have been developed into efficient vectors for delivering genome-engineering reagents. In addition to altering the host genome, these methods can target pathogens to engineer molecular immunity. Indeed, recent studies have shown that clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) systems that target the genomes of DNA viruses can interfere with viral activity and limit viral symptoms in planta, demonstrating the utility of this system for engineering molecular immunity in plants. CRISPR/Cas9 can efficiently target single and multiple viral infections and confer plant immunity. Here, we discuss the use of site-specific nucleases to engineer molecular immunity against DNA and RNA viruses in plants. We also explore how to address the potential challenges encountered when producing plants with engineered resistance to single and mixed viral infections.

CRISPR interference can prevent natural transformation and virulence acquisition during in vivo bacterial infection.

Science.gov (United States)

Bikard, David; Hatoum-Aslan, Asma; Mucida, Daniel; Marraffini, Luciano A

2012-08-16

Pathogenic bacterial strains emerge largely due to transfer of virulence and antimicrobial resistance genes between bacteria, a process known as horizontal gene transfer (HGT). Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci of bacteria and archaea encode a sequence-specific defense mechanism against bacteriophages and constitute a programmable barrier to HGT. However, the impact of CRISPRs on the emergence of virulence is unknown. We programmed the human pathogen Streptococcus pneumoniae with CRISPR sequences that target capsule genes, an essential pneumococcal virulence factor, and show that CRISPR interference can prevent transformation of nonencapsulated, avirulent pneumococci into capsulated, virulent strains during infection in mice. Further, at low frequencies bacteria can lose CRISPR function, acquire capsule genes, and mount a successful infection. These results demonstrate that CRISPR interference can prevent the emergence of virulence in vivo and that strong selective pressure for virulence or antibiotic resistance can lead to CRISPR loss in bacterial pathogens. Copyright © 2012 Elsevier Inc. All rights reserved.

CRISPR-Cas Technologies and Applications in Food Bacteria.

Science.gov (United States)

Stout, Emily; Klaenhammer, Todd; Barrangou, Rodolphe

2017-02-28

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins form adaptive immune systems that occur in many bacteria and most archaea. In addition to protecting bacteria from phages and other invasive mobile genetic elements, CRISPR-Cas molecular machines can be repurposed as tool kits for applications relevant to the food industry. A primary concern of the food industry has long been the proper management of food-related bacteria, with a focus on both enhancing the outcomes of beneficial microorganisms such as starter cultures and probiotics and limiting the presence of detrimental organisms such as pathogens and spoilage microorganisms. This review introduces CRISPR-Cas as a novel set of technologies to manage food bacteria and offers insights into CRISPR-Cas biology. It primarily focuses on the applications of CRISPR-Cas systems and tools in starter cultures and probiotics, encompassing strain-typing, phage resistance, plasmid vaccination, genome editing, and antimicrobial activity.

CrRNA-Protospacer Recognition during CRISPR- Directed DNA Interference Sulfolobus islandicus REY 15A and Structural Studies of CRISPR Binding Proteins (CBP) of Crenarchaeon Sulfolobus

DEFF Research Database (Denmark)

Mousaei, Marzieh

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats and associated proteins) is one of the important known immune mechanisms in archaea and bacteria. This adaptive immune system degrades invading genetic elements and protects the cell. Amongst 3 main types I, II and III...... of CRISPR system, two types (I and III) are found in archaea. However, in Sulfolobus species, subtypes IA, I-D, and III-B, III-D and rarely III-A are found. The model organism used for interference and structural studies is S. islandicus REY15A which carries subtypes I-A and III-B (α and β). Besides CRISPR...... ribonucleoprotein complex which is involved directly in defense, there are some less- known parts of the system including CPBs (CRISPR repeat-binding proteins) which are suggested to play a role in transcription. In the first part of my thesis, I provide a brief introduction to archaea and viruses that infect...

Application of CRISPR/Cas9 Technology to HBV

Directory of Open Access Journals (Sweden)

Guigao Lin

2015-11-01

Full Text Available More than 240 million people around the world are chronically infected with hepatitis B virus (HBV. Nucleos(tide analogs and interferon are the only two families of drugs to treat HBV currently. However, none of these anti-virals directly target the stable nuclear covalently closed circular DNA (cccDNA, which acts as a transcription template for viral mRNA and pre-genomic RNA synthesis and secures virus persistence. Thus, the fact that only a small number of patients treated achieve sustained viral response (SVR or cure, highlights the need for new therapies against HBV. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9 gene editing system can specifically target the conserved regions of the HBV genome. This results in robust viral suppression and provides a promising tool for eradicating the virus. In this review, we discuss the function and application of the CRISPR/Cas9 system as a novel therapy for HBV.

Modification of ethylene sensitivity in ornamental plants using CRISPR/Cas9

DEFF Research Database (Denmark)

Kemp, Oliver; Favero, Bruno Trevenzoli; Hegelund, Josefine Nymark

2017-01-01

; the Clustered Regularly Interspaced Palindromic Repeats (CRISPR) RNA guided Cas9 DNA nuclease (CRISPR/Cas9). CRISPR/Cas9 may be employed to introduce targeted double-stranded breaks (DSBs) at desired sites in the host genome. The DSBs will be repaired by the non-homologous end-joining (NHEJ) repair mechanism...... which often results in small indels and consequently gene knockout. The CRISPR/Cas9 system consists of a protein DNA nuclease (Cas9) which is guided to the target sequence by a small RNA molecule (sgRNA) that recognizes a 20 bp target sequence in the genome situated immediately downstream of a 3 bp...... protospacer adjacent motif (PAM). The sgRNA confers the sequence specificity of the CRISPR/Cas9 complex and may thus be designed to target virtually any sequence, a feature that has made it the method of choice within precise genetic engineering. Although most research with CRISPR/Cas9 has been conducted...

A Comprehensive Curation Shows the Dynamic Evolutionary Patterns of Prokaryotic CRISPRs

Directory of Open Access Journals (Sweden)

Guoqin Mai

2016-01-01

Full Text Available Motivation. Clustered regularly interspaced short palindromic repeat (CRISPR is a genetic element with active regulation roles for foreign invasive genes in the prokaryotic genomes and has been engineered to work with the CRISPR-associated sequence (Cas gene Cas9 as one of the modern genome editing technologies. Due to inconsistent definitions, the existing CRISPR detection programs seem to have missed some weak CRISPR signals. Results. This study manually curates all the currently annotated CRISPR elements in the prokaryotic genomes and proposes 95 updates to the annotations. A new definition is proposed to cover all the CRISPRs. The comprehensive comparison of CRISPR numbers on the taxonomic levels of both domains and genus shows high variations for closely related species even in the same genus. The detailed investigation of how CRISPRs are evolutionarily manipulated in the 8 completely sequenced species in the genus Thermoanaerobacter demonstrates that transposons act as a frequent tool for splitting long CRISPRs into shorter ones along a long evolutionary history.

Synthetic RNAs for Gene Regulation: Design Principles and Computational Tools

Energy Technology Data Exchange (ETDEWEB)

Laganà, Alessandro [Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, The Ohio State University, Columbus, OH (United States); Shasha, Dennis [Courant Institute of Mathematical Sciences, New York University, New York, NY (United States); Croce, Carlo Maria [Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, The Ohio State University, Columbus, OH (United States)

2014-12-11

The use of synthetic non-coding RNAs for post-transcriptional regulation of gene expression has not only become a standard laboratory tool for gene functional studies but it has also opened up new perspectives in the design of new and potentially promising therapeutic strategies. Bioinformatics has provided researchers with a variety of tools for the design, the analysis, and the evaluation of RNAi agents such as small-interfering RNA (siRNA), short-hairpin RNA (shRNA), artificial microRNA (a-miR), and microRNA sponges. More recently, a new system for genome engineering based on the bacterial CRISPR-Cas9 system (Clustered Regularly Interspaced Short Palindromic Repeats), was shown to have the potential to also regulate gene expression at both transcriptional and post-transcriptional level in a more specific way. In this mini review, we present RNAi and CRISPRi design principles and discuss the advantages and limitations of the current design approaches.

Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein

Directory of Open Access Journals (Sweden)

April Pawluk

2017-12-01

Full Text Available CRISPR (clustered regularly interspaced short palindromic repeat-Cas adaptive immune systems are prevalent defense mechanisms in bacteria and archaea. They provide sequence-specific detection and neutralization of foreign nucleic acids such as bacteriophages and plasmids. One mechanism by which phages and other mobile genetic elements are able to overcome the CRISPR-Cas system is through the expression of anti-CRISPR proteins. Over 20 different families of anti-CRISPR proteins have been described, each of which inhibits a particular type of CRISPR-Cas system. In this work, we determined the structure of type I-E anti-CRISPR protein AcrE1 by X-ray crystallography. We show that AcrE1 binds to the CRISPR-associated helicase/nuclease Cas3 and that the C-terminal region of the anti-CRISPR protein is important for its inhibitory activity. We further show that AcrE1 can convert the endogenous type I-E CRISPR system into a programmable transcriptional repressor.

Coevolution of CRISPR bacteria and phage in 2 dimensions

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Han, Pu; Deem, Michael

2014-03-01

CRISPR (cluster regularly interspaced short palindromic repeats) is a newly discovered adaptive, heritable immune system of prokaryotes. It can prevent infection of prokaryotes by phage. Most bacteria and almost all archae have CRISPR. The CRISPR system incorporates short nucleotide sequences from viruses. These incorporated sequences provide a historical record of the host and predator coevolution. We simulate the coevolution of bacteria and phage in 2 dimensions. Each phage has multiple proto-spacers that the bacteria can incorporate. Each bacterium can store multiple spacers in its CRISPR. Phages can escape recognition by the CRISPR system via point mutation or recombination. We will discuss the different evolutionary consequences of point mutation or recombination on the coevolution of bacteria and phage. We will also discuss an intriguing ``dynamic phase transition'' in the number of phage as a function of time and mutation rate. We will show that due to the arm race between phages and bacteria, the frequency of spacers and proto-spacers in a population can oscillate quite rapidly.

CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity

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Barrangou, Rodolphe; Marraffini, Luciano A.

2014-01-01

Summary Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), and associated proteins (Cas) comprise the CRISPR-Cas system, which confers adaptive immunity against exogenic elements in many bacteria and most archaea. CRISPR-mediated immunization occurs through the uptake of DNA from invasive genetic elements such as plasmids and viruses, followed by its integration into CRISPR loci. These loci are subsequently transcribed and processed into small interfering RNAs that guide nucleases for specific cleavage of complementary sequences. Conceptually, CRISPR-Cas shares functional features with the mammalian adaptive immune system, while also exhibiting characteristics of Lamarckian evolution. Because immune markers spliced from exogenous agents are integrated iteratively in CRISPR loci, they constitute a genetic record of vaccination events and reflect environmental conditions and changes over time. Cas endonucleases, which can be reprogrammed by small guide RNAs have shown unprecedented potential and flexibility for genome editing, and can be repurposed for numerous DNA targeting applications including transcriptional control. PMID:24766887

[Advances in CRISPR-Cas-mediated genome editing system in plants].

Science.gov (United States)

Wang, Chun; Wang, Kejian

2017-10-25

Targeted genome editing technology is an important tool to study the function of genes and to modify organisms at the genetic level. Recently, CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) system has emerged as an efficient tool for specific genome editing in animals and plants. CRISPR-Cas system uses CRISPR-associated endonuclease and a guide RNA to generate double-strand breaks at the target DNA site, subsequently leading to genetic modifications. CRISPR-Cas system has received widespread attention for manipulating the genomes with simple, easy and high specificity. This review summarizes recent advances of diverse applications of the CRISPR-Cas toolkit in plant research and crop breeding, including expanding the range of genome editing, precise editing of a target base, and efficient DNA-free genome editing technology. This review also discusses the potential challenges and application prospect in the future, and provides a useful reference for researchers who are interested in this field.

Targeted knock-in of an scFv-Fc antibody gene into the hprt locus of Chinese hamster ovary cells using CRISPR/Cas9 and CRIS-PITCh systems.

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Kawabe, Yoshinori; Komatsu, Shinya; Komatsu, Shodai; Murakami, Mai; Ito, Akira; Sakuma, Tetsushi; Nakamura, Takahiro; Yamamoto, Takashi; Kamihira, Masamichi

2018-05-01

Chinese hamster ovary (CHO) cells have been used as host cells for the production of pharmaceutical proteins. For the high and stable production of target proteins, the transgene should be integrated into a suitable genomic locus of host cells. Here, we generated knock-in CHO cells, in which transgene cassettes without a vector backbone sequence were integrated into the hprt locus of the CHO genome using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) systems. We investigated the efficiency of targeted knock-in of transgenes using these systems. As a practical example, we generated knock-in CHO cells producing an scFv-Fc antibody using the CRIS-PITCh system mediated by microhomology sequences for targeting. We found that the CRIS-PITCh system can facilitate targeted knock-in for CHO cell engineering. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency.

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Song, Jun; Yang, Dongshan; Xu, Jie; Zhu, Tianqing; Chen, Y Eugene; Zhang, Jifeng

2016-01-28

Zinc-finger nuclease, transcription activator-like effector nuclease and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) are becoming major tools for genome editing. Importantly, knock-in in several non-rodent species has been finally achieved thanks to these customizable nucleases; yet the rates remain to be further improved. We hypothesize that inhibiting non-homologous end joining (NHEJ) or enhancing homology-directed repair (HDR) will improve the nuclease-mediated knock-in efficiency. Here we show that the in vitro application of an HDR enhancer, RS-1, increases the knock-in efficiency by two- to five-fold at different loci, whereas NHEJ inhibitor SCR7 has minimal effects. We then apply RS-1 for animal production and have achieved multifold improvement on the knock-in rates as well. Our work presents tools to nuclease-mediated knock-in animal production, and sheds light on improving gene-targeting efficiencies on pluripotent stem cells.

CRISPR-cas System as a Genome Engineering Platform: Applications in Biomedicine and Biotechnology.

Science.gov (United States)

Hashemi, Atieh

2018-01-01

Genome editing mediated by Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its associated proteins (Cas) has recently been considered to be used as efficient, rapid and site-specific tool in the modification of endogenous genes in biomedically important cell types and whole organisms. It has become a predictable and precise method of choice for genome engineering by specifying a 20-nt targeting sequence within its guide RNA. Firstly, this review aims to describe the biology of CRISPR system. Next, the applications of CRISPR-Cas9 in various ways, such as efficient generation of a wide variety of biomedically important cellular models as well as those of animals, modifying epigenomes, conducting genome-wide screens, gene therapy, labelling specific genomic loci in living cells, metabolic engineering of yeast and bacteria and endogenous gene expression regulation by an altered version of this system were reviewed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Deciphering, Communicating, and Engineering the CRISPR PAM.

Science.gov (United States)

Leenay, Ryan T; Beisel, Chase L

2017-01-20

Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their flanking CRISPR-associated (cas) genes make up RNA-guided, adaptive immune systems in prokaryotes whose effector proteins have become powerful tools for basic research and biotechnology. While the Cas effector proteins are remarkably diverse, they commonly rely on protospacer-adjacent motifs (PAMs) as the first step in target recognition. PAM sequences are known to vary considerably between systems and have proven to be difficult to predict, spurring the need for new tools to rapidly identify and communicate these sequences. Recent advances have also shown that Cas proteins can be engineered to alter PAM recognition, opening new opportunities to develop CRISPR-based tools with enhanced targeting capabilities. In this review, we discuss the properties of the CRISPR PAM and the emerging tools for determining, visualizing, and engineering PAM recognition. We also propose a standard means of orienting the PAM to simplify how its location and sequence are communicated. Copyright © 2016 Elsevier Ltd. All rights reserved.

RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency

Science.gov (United States)

Song, Jun; Yang, Dongshan; Xu, Jie; Zhu, Tianqing; Chen, Y. Eugene; Zhang, Jifeng

2016-01-01

Zinc-finger nuclease, transcription activator-like effector nuclease and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) are becoming major tools for genome editing. Importantly, knock-in in several non-rodent species has been finally achieved thanks to these customizable nucleases; yet the rates remain to be further improved. We hypothesize that inhibiting non-homologous end joining (NHEJ) or enhancing homology-directed repair (HDR) will improve the nuclease-mediated knock-in efficiency. Here we show that the in vitro application of an HDR enhancer, RS-1, increases the knock-in efficiency by two- to five-fold at different loci, whereas NHEJ inhibitor SCR7 has minimal effects. We then apply RS-1 for animal production and have achieved multifold improvement on the knock-in rates as well. Our work presents tools to nuclease-mediated knock-in animal production, and sheds light on improving gene-targeting efficiencies on pluripotent stem cells. PMID:26817820

CRISPR Technology Reveals RAD(51)-ical Mechanisms of Repair in Roundworms: An Educational Primer for Use with "Promotion of Homologous Recombination by SWS-1 in Complex with RAD-51 Paralogs in Caenorhabditis elegans".

Science.gov (United States)

Turcotte, Carolyn A; Andrews, Nicolas P; Sloat, Solomon A; Checchi, Paula M

2016-11-01

The mechanisms cells use to maintain genetic fidelity via DNA repair and the accuracy of these processes have garnered interest from scientists engaged in basic research to clinicians seeking improved treatment for cancer patients. Despite the continued advances, many details of DNA repair are still incompletely understood. In addition, the inherent complexity of DNA repair processes, even at the most fundamental level, makes it a challenging topic. This primer is meant to assist both educators and students in using a recent paper, "Promotion of homologous recombination by SWS-1 in complex with RAD-51 paralogs in Caenorhabditis elegans," to understand mechanisms of DNA repair. The goals of this primer are to highlight and clarify several key techniques utilized, with special emphasis on the clustered, regularly interspaced, short palindromic repeats technique and the ways in which it has revolutionized genetics research, as well as to provide questions for deeper in-class discussion. Copyright © 2016 by the Genetics Society of America.

Tropomyosin 2 heterozygous knockout in mice using CRISPR-Cas9 system displays the inhibition of injury-induced epithelial-mesenchymal transition, and lens opacity

Science.gov (United States)

Shibata, Teppei; Shibata, Shinsuke; Ishigaki, Yasuhito; Kiyokawa, Etsuko; Ikawa, Masahito; Singh, Dhirendra P.; Sasaki, Hiroshi; Kubo, Eri

2018-01-01

The process of epithelial–mesenchymal transition (EMT) of lens epithelial cells (LECs) after cataract surgery contributes to tissue fibrosis, wound healing and lens regeneration via a mechanism not yet fully understood. Here, we show that tropomyosin 2 (Tpm2) plays a critical role in wound healing and lens aging. Posterior capsular opacification (PCO) after lens extraction surgery was accompanied by elevated expression of Tpm2. Tpm2 heterozygous knockout mice, generated via the clustered regularly interspaced short palindromic repeat/ Cas9 (CRISPR/Cas9) system showed promoted progression of cataract with age. Further, injury-induced EMT of the mouse lens epithelium, as evaluated histologically and by the expression patterns of Tpm1 and Tpm2, was attenuated in the absence of Tpm2. In conclusion, Tpm2 may be important in maintaining lens physiology and morphology. However, Tpm2 is involved in the progression of EMT during the wound healing process of mouse LECs, suggesting that inhibition of Tpm2 may suppress PCO. PMID:29510160

Design of a CRISPR-Cas system to increase resistance of Bacillus subtilis to bacteriophage SPP1.

Science.gov (United States)

Jakutyte-Giraitiene, Lina; Gasiunas, Giedrius

2016-08-01

Clustered regularly interspaced short palindromic repeats (CRISPR) together with CRISPR-associated (cas) genes form an adaptive prokaryotic immune system which provides acquired resistance against viruses and plasmids. Bacillus subtilis presently is the best-characterized laboratory model for Gram-positive bacteria and also widely used for industrial production of enzymes, vitamins and antibiotics. In this study, we show that type II-A CRISPR-Cas system from Streptococcus thermophilus can be transferred into B. subtilis and provides heterologous protection against phage infection. We engineered a heterologous host by cloning S. thermophilus Cas9 and a spacer targeting bacteriophage SPP1 into the chromosome of B. subtilis, which does not harbor its own CRISPR-Cas systems. We found that the heterologous CRISPR-Cas system is functionally active in B. subtilis and provides resistance against bacteriophage SPP1 infection. The high efficiency of the acquired immunity against phage could be useful in generation of biotechnologically important B. subtilis strains with engineered chromosomes.

Reduced Drought Tolerance by CRISPR/Cas9-Mediated SlMAPK3 Mutagenesis in Tomato Plants.

Science.gov (United States)

Wang, Liu; Chen, Lin; Li, Rui; Zhao, Ruirui; Yang, Meijing; Sheng, Jiping; Shen, Lin

2017-10-04

Drought stress is one of the most destructive environmental factors that affect tomato plants adversely. Mitogen-activated protein kinases (MAPKs) are important signaling molecules that respond to drought stress. In this study, SlMAPK3 was induced by drought stress, and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system was utilized to generate slmapk3 mutants. Two independent T1 transgenic lines and wild-type (WT) tomato plants were used for analysis of drought tolerance. Compared with WT plants, slmapk3 mutants exhibited more severe wilting symptom, higher hydrogen peroxide content, lower antioxidant enzymes activities, and suffered more membrane damage under drought stress. Furthermore, knockout of SlMAPK3 led to up- or down-regulated expressions of drought stress-responsive genes including SlLOX, SlGST, and SlDREB. The results suggest that SlMAPK3 is involved in drought response in tomato plants by protecting cell membranes from oxidative damage and modulating transcription of stress-related genes.

Abundance and distribution of the highly iterated palindrome 1 (HIP1) among prokaryotes

OpenAIRE

Delaye, Luis; Moya, Andrés

2011-01-01

We have studied the abundance and phylogenetic distribution of the Highly Iterated Palindrome 1 (HIP1) among sequenced prokaryotic genomes. We show that an overrepresentation of HIP1 is exclusive of some lineages of cyanobacteria, and that this abundance was gained only once during evolution and was subsequently lost in the lineage leading to marine pico-cyanobacteria. We show that among cyanobacterial protein sequences with annotated Pfam domains, only OpcA (glucose 6-phosphate dehydrogenase...

Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-coupled Affinity Purification/Mass Spectrometry Analysis Revealed a Novel Role of Neurofibromin in mTOR Signaling.

Science.gov (United States)

Li, Xu; Gao, Min; Choi, Jong Min; Kim, Beom-Jun; Zhou, Mao-Tian; Chen, Zhen; Jain, Antrix N; Jung, Sung Yun; Yuan, Jingsong; Wang, Wenqi; Wang, Yi; Chen, Junjie

2017-04-01

Neurofibromin (NF1) is a well known tumor suppressor that is commonly mutated in cancer patients. It physically interacts with RAS and negatively regulates RAS GTPase activity. Despite the importance of NF1 in cancer, a high quality endogenous NF1 interactome has yet to be established. In this study, we combined c lustered, r egularly i nterspaced s hort p alindromic r epeats (CRISPR)/Cas9-mediated gene knock-out technology with affinity purification using antibodies against endogenous proteins, followed by mass spectrometry analysis, to sensitively and accurately detect NF1 protein-protein interactions in unaltered in vivo settings. Using this system, we analyzed endogenous NF1-associated protein complexes and identified 49 high-confidence candidate interaction proteins, including RAS and other functionally relevant proteins. Through functional validation, we found that NF1 negatively regulates mechanistic target of rapamycin signaling (mTOR) in a LAMTOR1-dependent manner. In addition, the cell growth and survival of NF1-deficient cells have become dependent on hyperactivation of the mTOR pathway, and the tumorigenic properties of these cells have become dependent on LAMTOR1. Taken together, our findings may provide novel insights into therapeutic approaches targeting NF1-deficient tumors. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Optimal packaging of FIV genomic RNA depends upon a conserved long-range interaction and a palindromic sequence within gag.

Science.gov (United States)

Rizvi, Tahir A; Kenyon, Julia C; Ali, Jahabar; Aktar, Suriya J; Phillip, Pretty S; Ghazawi, Akela; Mustafa, Farah; Lever, Andrew M L

2010-10-15

The feline immunodeficiency virus (FIV) is a lentivirus that is related to human immunodeficiency virus (HIV), causing a similar pathology in cats. It is a potential small animal model for AIDS and the FIV-based vectors are also being pursued for human gene therapy. Previous studies have mapped the FIV packaging signal (ψ) to two or more discontinuous regions within the 5' 511 nt of the genomic RNA and structural analyses have determined its secondary structure. The 5' and 3' sequences within ψ region interact through extensive long-range interactions (LRIs), including a conserved heptanucleotide interaction between R/U5 and gag. Other secondary structural elements identified include a conserved 150 nt stem-loop (SL2) and a small palindromic stem-loop within gag open reading frame that might act as a viral dimerization initiation site. We have performed extensive mutational analysis of these sequences and structures and ascertained their importance in FIV packaging using a trans-complementation assay. Disrupting the conserved heptanucleotide LRI to prevent base pairing between R/U5 and gag reduced packaging by 2.8-5.5 fold. Restoration of pairing using an alternative, non-wild type (wt) LRI sequence restored RNA packaging and propagation to wt levels, suggesting that it is the structure of the LRI, rather than its sequence, that is important for FIV packaging. Disrupting the palindrome within gag reduced packaging by 1.5-3-fold, but substitution with a different palindromic sequence did not restore packaging completely, suggesting that the sequence of this region as well as its palindromic nature is important. Mutation of individual regions of SL2 did not have a pronounced effect on FIV packaging, suggesting that either it is the structure of SL2 as a whole that is necessary for optimal packaging, or that there is redundancy within this structure. The mutational analysis presented here has further validated the previously predicted RNA secondary structure of FIV

The dyad palindromic glutathione transferase P enhancer binds multiple factors including AP1.

OpenAIRE

Diccianni, M B; Imagawa, M; Muramatsu, M

1992-01-01

Glutathione Transferase P (GST-P) gene expression is dominantly regulated by an upstream enhancer (GPEI) consisting of a dyad of palindromically oriented imperfect TPA (12-O-tetradecanoyl-phorbol-13-acetate)-responsive elements (TRE). GPEI is active in AP1-lacking F9 cells as well in AP1-containing HeLa cells. Despite GPEI's similarity to a TRE, c-jun co-transfection has only a minimal effect on transactivation. Antisense c-jun and c-fos co-transfection experiments further demonstrate the lac...

CRISPR and personalized Treg therapy: new insights into the treatment of rheumatoid arthritis.

Science.gov (United States)

Safari, Fatemeh; Farajnia, Safar; Arya, Maryam; Zarredar, Habib; Nasrolahi, Ava

2018-06-01

Rheumatoid arthritis (RA), as one of the most disabling autoimmune diseases, is a common health problem that progressively reduces the life quality of patients. Although various biologics have been introduced for RA, attempts to establish an efficient long-term therapies failed due to the heterogeneity of this disease. In the last decade, immunomodulatory approaches such as T cell adoptive therapy have been developed for controlling autoimmunity. Regulatory T cells (Tregs), the major self-tolerance mediator, are crucial for down-regulation of aberrant immune stimulations. Hence, recruiting ex vivo Tregs emerged as a promising therapy for a variety of autoimmune diseases. The major bottleneck of the Treg adoptive therapy is maintaining the in vivo stability and plasticity of these fascinating cells. Recent progress in genome editing technology clustered regularly interspaced short palindromic repeats (CRISPR) in combination with CRISPR-associated (Cas) 9 system provided a new solution for this bottleneck. The present paper discusses RA pathogenesis and the potential application of new developments in CRISPR-mediated Treg genome editing in personalized therapy of RA.

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.

Science.gov (United States)

Sternberg, Samuel H; Redding, Sy; Jinek, Martin; Greene, Eric C; Doudna, Jennifer A

2014-03-06

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

Editing plants for virus resistance using CRISPR-Cas.

Science.gov (United States)

Green, J C; Hu, J S

This minireview summarizes recent advancements using the clustered regularly interspaced palindromic repeats-associated nuclease systems (CRISPR-Cas) derived from prokaryotes to breed plants resistant to DNA and RNA viruses. The CRISPR-Cas system represents a powerful tool able to edit and insert novel traits into plants precisely at chosen loci offering enormous advantages to classical breeding. Approaches to engineering plant virus resistance in both transgenic and non-transgenic plants are discussed. Iterations of the CRISPR-Cas system, FnCas9 and C2c2 capable of editing RNA in eukaryotic cells offer a particular advantage for providing resistance to RNA viruses which represent the great majority of known plant viruses. Scientists have obtained conflicting results using gene silencing technology to produce transgenic plants resistant to geminiviruses. CRISPR-Cas systems engineered in plants to target geminiviruses have consistently reduced virus accumulation providing increased resistance to virus infection. CRISPR-Cas may provide novel and reliable approaches to control geminiviruses and other ssDNA viruses such as Banana bunchy top virus (BBTV).

Genome editing in plants: Advancing crop transformation and overview of tools.

Science.gov (United States)

Shah, Tariq; Andleeb, Tayyaba; Lateef, Sadia; Noor, Mehmood Ali

2018-05-07

Genome manipulation technology is one of emerging field which brings real revolution in genetic engineering and biotechnology. Targeted editing of genomes pave path to address a wide range of goals not only to improve quality and productivity of crops but also permit to investigate the fundamental roots of biological systems. These goals includes creation of plants with valued compositional properties and with characters that confer resistance to numerous biotic and abiotic stresses. Numerous novel genome editing systems have been introduced during the past few years; these comprise zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9). Genome editing technique is consistent for improving average yield to achieve the growing demands of the world's existing food famine and to launch a feasible and environmentally safe agriculture scheme, to more specific, productive, cost-effective and eco-friendly. These exciting novel methods, concisely reviewed herein, have verified themselves as efficient and reliable tools for the genetic improvement of plants. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Genome Editing in Sugarcane: Challenges ahead

Directory of Open Access Journals (Sweden)

Chakravarthi Mohan

2016-10-01

Full Text Available Genome editing opens new and unique opportunities for researchers to enhance crop production. Until 2013, the zinc finger nucleases (ZFNs and transcription activator-like effector nucleases (TALENs were the key tools used for genome editing applications. The advent of RNA-guided engineered nucleases - the type II clustered regularly interspaced short palindromic repeat (CRISPR/Cas9 (CRISPR-associated system from Streptococcus pyogenes holds great potential since it is simple, effective and more versatile than ZFNs and TALENs. CRISPR/Cas9 system has already been successfully employed in several crop plants. Use of these techniques is in its infant stage in sugarcane. Jung and Altpeter (2016 have reported TALEN mediated approach for the first time to reduce lignin content in sugarcane to make it amenable for biofuel production. This is so far the only report describing genome editing in sugarcane. Large genome size, polyploidy, low transformation efficiency, transgene silencing and lack of high throughput screening techniques are certainly great challenges for genome editing in sugarcane which would be discussed in detail in this review.

Efficient Genome Editing in the Oomycete Phytophthora sojae Using CRISPR/Cas9.

Science.gov (United States)

Fang, Yufeng; Cui, Linkai; Gu, Biao; Arredondo, Felipe; Tyler, Brett M

2017-02-06

Phytophthora is a filamentous fungus-like microorganism, but belongs to the oomycetes, in the kingdom Stramenopila. Phytophthora species are notorious as plant destroyers, causing multibillion-dollar damage to agriculture and natural ecosystems worldwide annually. For a long time, genome editing has been unattainable in oomycetes, because of their extremely low rate of homologous recombination. The recent implementation of the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system in the soybean pathogen Phytophthora sojae, an experimental model for oomycetes, has opened up a powerful new research capability for the oomycete community. Here, we describe a detailed protocol for CRISPR/Cas9-mediated genome editing in P. sojae, including single guide RNA (sgRNA) design and construction, efficient gene replacement, and mutant-screening strategies. This protocol should be generally applicable for most culturable oomycetes. We also describe an optimized transformation method that is useful for other Phytophthora spp. including P. capsici and P. parasitica. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration

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Suzuki, Keiichiro

2016-11-15

Targeted genome editing via engineered nucleases is an exciting area of biomedical research and holds potential for clinical applications. Despite rapid advances in the field, in vivo targeted transgene integration is still infeasible because current tools are inefficient1, especially for non-dividing cells, which compose most adult tissues. This poses a barrier for uncovering fundamental biological principles and developing treatments for a broad range of genetic disorders2. Based on clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9)3, 4 technology, here we devise a homology-independent targeted integration (HITI) strategy, which allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and, more importantly, in vivo (for example, in neurons of postnatal mammals). As a proof of concept of its therapeutic potential, we demonstrate the efficacy of HITI in improving visual function using a rat model of the retinal degeneration condition retinitis pigmentosa. The HITI method presented here establishes new avenues for basic research and targeted gene therapies.

Fanconi anemia gene editing by the CRISPR/Cas9 system.

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Osborn, Mark J; Gabriel, Richard; Webber, Beau R; DeFeo, Anthony P; McElroy, Amber N; Jarjour, Jordan; Starker, Colby G; Wagner, John E; Joung, J Keith; Voytas, Daniel F; von Kalle, Christof; Schmidt, Manfred; Blazar, Bruce R; Tolar, Jakub

2015-02-01

Genome engineering with designer nucleases is a rapidly progressing field, and the ability to correct human gene mutations in situ is highly desirable. We employed fibroblasts derived from a patient with Fanconi anemia as a model to test the ability of the clustered regularly interspaced short palindromic repeats/Cas9 nuclease system to mediate gene correction. We show that the Cas9 nuclease and nickase each resulted in gene correction, but the nickase, because of its ability to preferentially mediate homology-directed repair, resulted in a higher frequency of corrected clonal isolates. To assess the off-target effects, we used both a predictive software platform to identify intragenic sequences of homology as well as a genome-wide screen utilizing linear amplification-mediated PCR. We observed no off-target activity and show RNA-guided endonuclease candidate sites that do not possess low sequence complexity function in a highly specific manner. Collectively, we provide proof of principle for precision genome editing in Fanconi anemia, a DNA repair-deficient human disorder.

Genome-wide Specificity of Highly Efficient TALENs and CRISPR/Cas9 for T Cell Receptor Modification

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Friederike Knipping

2017-03-01

Full Text Available In T cells with transgenic high-avidity T cell receptors (TCRs, endogenous and transferred TCR chains compete for surface expression and may pair inappropriately, potentially causing autoimmunity. To knock out endogenous TCR expression, we assembled 12 transcription activator-like effector nucleases (TALENs and five guide RNAs (gRNAs from the clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR-associated (Cas9 system. Using TALEN mRNA, TCR knockout was successful in up to 81% of T cells. Additionally, we were able to verify targeted gene addition of a GFP gene by homology-directed repair at the TALEN target site, using a donor suitable for replacement of the reporter transgene with therapeutic TCR chains. Remarkably, analysis of TALEN and CRISPR/Cas9 specificity using integrase-defective lentiviral vector capture revealed only one off-target site for one of the gRNAs and three off-target sites for both of the TALENs, indicating a high level of specificity. Collectively, our work shows highly efficient and specific nucleases for T cell engineering.

CRISPR/Cas9 nuclease-mediated gene knock-in in bovine-induced pluripotent cells.

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Heo, Young Tae; Quan, Xiaoyuan; Xu, Yong Nan; Baek, Soonbong; Choi, Hwan; Kim, Nam-Hyung; Kim, Jongpil

2015-02-01

Efficient and precise genetic engineering in livestock such as cattle holds great promise in agriculture and biomedicine. However, techniques that generate pluripotent stem cells, as well as reliable tools for gene targeting in livestock, are still inefficient, and thus not routinely used. Here, we report highly efficient gene targeting in the bovine genome using bovine pluripotent cells and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease. First, we generate induced pluripotent stem cells (iPSCs) from bovine somatic fibroblasts by the ectopic expression of yamanaka factors and GSK3β and MEK inhibitor (2i) treatment. We observed that these bovine iPSCs are highly similar to naïve pluripotent stem cells with regard to gene expression and developmental potential in teratomas. Moreover, CRISPR/Cas9 nuclease, which was specific for the bovine NANOG locus, showed highly efficient editing of the bovine genome in bovine iPSCs and embryos. To conclude, CRISPR/Cas9 nuclease-mediated homologous recombination targeting in bovine pluripotent cells is an efficient gene editing method that can be used to generate transgenic livestock in the future.

CRISPR/Cas9-Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells.

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Sharma, Arun; Toepfer, Christopher N; Ward, Tarsha; Wasson, Lauren; Agarwal, Radhika; Conner, David A; Hu, Johnny H; Seidman, Christine E

2018-01-24

Human induced pluripotent stem cells (hiPSCs) can be used to mass produce surrogates of human tissues, enabling new advances in drug screening, disease modeling, and cell therapy. Recent developments in clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing technology use homology-directed repair (HDR) to efficiently generate custom hiPSC lines harboring a variety of genomic insertions and deletions. Thus, hiPSCs that encode an endogenous protein fused to a fluorescent reporter protein can be rapidly created by employing CRISPR/Cas9 genome editing, enhancing HDR efficiency and optimizing homology arm length. These fluorescently tagged hiPSCs can be used to visualize protein function and dynamics in real time as cells proliferate and differentiate. Given that nearly any intracellular protein can be fluorescently tagged, this system serves as a powerful tool to facilitate new discoveries across many biological disciplines. In this unit, we present protocols for the design, generation, and monoclonal expansion of genetically customized hiPSCs encoding fluorescently tagged endogenous proteins. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

May I Cut in? Gene Editing Approaches in Human Induced Pluripotent Stem Cells.

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Brookhouser, Nicholas; Raman, Sreedevi; Potts, Christopher; Brafman, David A

2017-02-06

In the decade since Yamanaka and colleagues described methods to reprogram somatic cells into a pluripotent state, human induced pluripotent stem cells (hiPSCs) have demonstrated tremendous promise in numerous disease modeling, drug discovery, and regenerative medicine applications. More recently, the development and refinement of advanced gene transduction and editing technologies have further accelerated the potential of hiPSCs. In this review, we discuss the various gene editing technologies that are being implemented with hiPSCs. Specifically, we describe the emergence of technologies including zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 that can be used to edit the genome at precise locations, and discuss the strengths and weaknesses of each of these technologies. In addition, we present the current applications of these technologies in elucidating the mechanisms of human development and disease, developing novel and effective therapeutic molecules, and engineering cell-based therapies. Finally, we discuss the emerging technological advances in targeted gene editing methods.

CRISPR/Cas9 Genome Editing: A Promising Tool for Therapeutic Applications of Induced Pluripotent Stem Cells.

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Zhang, Yanli; Sastre, Danuta; Wang, Feng

2018-01-01

Induced pluripotent stem cells hold tremendous potential for biological and therapeutic applications. The development of efficient technologies for targeted genome alteration of stem cells in disease models is a prerequisite for utilizing stem cells to their full potential. The revolutionary technology for genome editing known as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) system is recently recognized as a powerful tool for editing DNA at specific loci. The ease of use of the CRISPR-Cas9 technology will allow us to improve our understanding of genomic variation in disease processes via cellular and animal models. More recently, this system was modified to repress (CRISPR interference, CRISPRi) or activate (CRISPR activation, CRISPRa) gene expression without alterations in the DNA, which amplified the scope of applications of CRISPR systems for stem cell biology. Here, we highlight latest advances of CRISPR-associated applications in human pluripotent stem cells. The challenges and future prospects of CRISPR-based systems for human research are also discussed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation confers resistance to HIV infection.

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Ye, Lin; Wang, Jiaming; Beyer, Ashley I; Teque, Fernando; Cradick, Thomas J; Qi, Zhongxia; Chang, Judy C; Bao, Gang; Muench, Marcus O; Yu, Jingwei; Levy, Jay A; Kan, Yuet Wai

2014-07-01

Individuals homozygous for the C-C chemokine receptor type 5 gene with 32-bp deletions (CCR5Δ32) are resistant to HIV-1 infection. In this study, we generated induced pluripotent stem cells (iPSCs) homozygous for the naturally occurring CCR5Δ32 mutation through genome editing of wild-type iPSCs using a combination of transcription activator-like effector nucleases (TALENs) or RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 together with the piggyBac technology. Remarkably, TALENs or CRISPR-Cas9-mediated double-strand DNA breaks resulted in up to 100% targeting of the colonies on one allele of which biallelic targeting occurred at an average of 14% with TALENs and 33% with CRISPR. Excision of the piggyBac using transposase seamlessly reproduced exactly the naturally occurring CCR5Δ32 mutation without detectable exogenous sequences. We differentiated these modified iPSCs into monocytes/macrophages and demonstrated their resistance to HIV-1 challenge. We propose that this strategy may provide an approach toward a functional cure of HIV-1 infection.

Diverse Genotypes of Yersinia pestis Caused Plague in Madagascar in 2007.

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Riehm, Julia M; Projahn, Michaela; Vogler, Amy J; Rajerison, Minoaerisoa; Andersen, Genevieve; Hall, Carina M; Zimmermann, Thomas; Soanandrasana, Rahelinirina; Andrianaivoarimanana, Voahangy; Straubinger, Reinhard K; Nottingham, Roxanne; Keim, Paul; Wagner, David M; Scholz, Holger C

2015-06-01

Yersinia pestis is the causative agent of human plague and is endemic in various African, Asian and American countries. In Madagascar, the disease represents a significant public health problem with hundreds of human cases a year. Unfortunately, poor infrastructure makes outbreak investigations challenging. DNA was extracted directly from 93 clinical samples from patients with a clinical diagnosis of plague in Madagascar in 2007. The extracted DNAs were then genotyped using three molecular genotyping methods, including, single nucleotide polymorphism (SNP) typing, multi-locus variable-number tandem repeat analysis (MLVA), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) analysis. These methods provided increasing resolution, respectively. The results of these analyses revealed that, in 2007, ten molecular groups, two newly described here and eight previously identified, were responsible for causing human plague in geographically distinct areas of Madagascar. Plague in Madagascar is caused by numerous distinct types of Y. pestis. Genotyping method choice should be based upon the discriminatory power needed, expense, and available data for any desired comparisons. We conclude that genotyping should be a standard tool used in epidemiological investigations of plague outbreaks.

Development and potential applications of CRISPR-Cas9 genome editing technology in sarcoma.

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Liu, Tang; Shen, Jacson K; Li, Zhihong; Choy, Edwin; Hornicek, Francis J; Duan, Zhenfeng

2016-04-01

Sarcomas include some of the most aggressive tumors and typically respond poorly to chemotherapy. In recent years, specific gene fusion/mutations and gene over-expression/activation have been shown to drive sarcoma pathogenesis and development. These emerging genomic alterations may provide targets for novel therapeutic strategies and have the potential to transform sarcoma patient care. The RNA-guided nuclease CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein-9 nuclease) is a convenient and versatile platform for site-specific genome editing and epigenome targeted modulation. Given that sarcoma is believed to develop as a result of genetic alterations in mesenchymal progenitor/stem cells, CRISPR-Cas9 genome editing technologies hold extensive application potentials in sarcoma models and therapies. We review the development and mechanisms of the CRISPR-Cas9 system in genome editing and introduce its application in sarcoma research and potential therapy in clinic. Additionally, we propose future directions and discuss the challenges faced with these applications, providing concise and enlightening information for readers interested in this area. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

CRISPR-Mediated Genomic Deletion of Sox2 in the Axolotl Shows a Requirement in Spinal Cord Neural Stem Cell Amplification during Tail Regeneration

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Ji-Feng Fei

2014-09-01

Full Text Available The salamander is the only tetrapod that functionally regenerates all cell types of the limb and spinal cord (SC and thus represents an important regeneration model, but the lack of gene-knockout technology has limited molecular analysis. We compared transcriptional activator-like effector nucleases (TALENs and clustered regularly interspaced short palindromic repeats (CRISPRs in the knockout of three loci in the axolotl and find that CRISPRs show highly penetrant knockout with less toxic effects compared to TALENs. Deletion of Sox2 in up to 100% of cells yielded viable F0 larvae with normal SC organization and ependymoglial cell marker expression such as GFAP and ZO-1. However, upon tail amputation, neural stem cell proliferation was inhibited, resulting in spinal-cord-specific regeneration failure. In contrast, the mesodermal blastema formed normally. Sox3 expression during development, but not regeneration, most likely allowed embryonic survival and the regeneration-specific phenotype. This analysis represents the first tissue-specific regeneration phenotype from the genomic deletion of a gene in the axolotl.

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

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Sternberg, Samuel H.; Redding, Sy; Jinek, Martin; Greene, Eric C.; Doudna, Jennifer A.

2014-03-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes.

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Ehrke-Schulz, Eric; Schiwon, Maren; Leitner, Theo; Dávid, Stephan; Bergmann, Thorsten; Liu, Jing; Ehrhardt, Anja

2017-12-07

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system revolutionized the field of gene editing but viral delivery of the CRISPR/Cas9 system has not been fully explored. Here we adapted clinically relevant high-capacity adenoviral vectors (HCAdV) devoid of all viral genes for the delivery of the CRISPR/Cas9 machinery using a single viral vector. We present a platform enabling fast transfer of the Cas9 gene and gRNA expression units into the HCAdV genome including the option to choose between constitutive or inducible Cas9 expression and gRNA multiplexing. Efficacy and versatility of this pipeline was exemplified by producing different CRISPR/Cas9-HCAdV targeting the human papillomavirus (HPV) 18 oncogene E6, the dystrophin gene causing Duchenne muscular dystrophy (DMD) and the HIV co-receptor C-C chemokine receptor type 5 (CCR5). All CRISPR/Cas9-HCAdV proved to be efficient to deliver the respective CRISPR/Cas9 expression units and to introduce the desired DNA double strand breaks at their intended target sites in immortalized and primary cells.

Genome Editing for Cancer Therapy: Delivery of Cas9 Protein/sgRNA Plasmid via a Gold Nanocluster/Lipid Core-Shell Nanocarrier.

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Wang, Peng; Zhang, Lingmin; Xie, Yangzhouyun; Wang, Nuoxin; Tang, Rongbing; Zheng, Wenfu; Jiang, Xingyu

2017-11-01

The type II bacterial clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 (CRISPR-associated protein) system (CRISPR-Cas9) is a powerful toolbox for gene-editing, however, the nonviral delivery of CRISPR-Cas9 to cells or tissues remains a key challenge. This paper reports a strategy to deliver Cas9 protein and single guide RNA (sgRNA) plasmid by a nanocarrier with a core of gold nanoclusters (GNs) and a shell of lipids. By modifying the GNs with HIV-1-transactivator of transcription peptide, the cargo (Cas9/sgRNA) can be delivered into cell nuclei. This strategy is utilized to treat melanoma by designing sgRNA targeting Polo-like kinase-1 ( Plk1 ) of the tumor. The nanoparticle (polyethylene glycol-lipid/GNs/Cas9 protein/sgPlk1 plasmid, LGCP) leads to >70% down-regulation of Plk1 protein expression of A375 cells in vitro. Moreover, the LGCP suppresses melanoma progress by 75% on mice. Thus, this strategy can deliver protein-nucleic acid hybrid agents for gene therapy.

CRISPR/Cas9-mediated noncoding RNA editing in human cancers.

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Yang, Jie; Meng, Xiaodan; Pan, Jinchang; Jiang, Nan; Zhou, Chengwei; Wu, Zhenhua; Gong, Zhaohui

2018-01-02

Cancer is characterized by multiple genetic and epigenetic alterations, including a higher prevalence of mutations of oncogenes and/or tumor suppressors. Mounting evidences have shown that noncoding RNAs (ncRNAs) are involved in the epigenetic regulation of cancer genes and their associated pathways. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (CRISPR/Cas9) system, a revolutionary genome-editing technology, has shed light on ncRNA-based cancer therapy. Here, we briefly introduce the classifications and mechanisms of CRISPR/Cas9 system. Importantly, we mainly focused on the applications of CRISPR/Cas9 system as a molecular tool for ncRNA (microRNA, long noncoding RNA and circular RNA, etc.) editing in human cancers, and the novel techniques that are based on CRISPR/Cas9 system. Additionally, the off-target effects and the corresponding solutions as well as the challenges toward CRISPR/Cas9 were also evaluated and discussed. Long- and short-ncRNAs have been employed as targets in precision oncology, and CRISPR/Cas9-mediated ncRNA editing may provide an excellent way to cure cancer.

Applications of Gene Editing Technologies to Cellular Therapies.

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Rein, Lindsay A M; Yang, Haeyoon; Chao, Nelson J

2018-03-27

Hematologic malignancies are characterized by genetic heterogeneity, making classic gene therapy with a goal of correcting 1 genetic defect ineffective in many of these diseases. Despite initial tribulations, gene therapy, as a field, has grown by leaps and bounds with the recent development of gene editing techniques including zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR) sequences and CRISPR-associated protein-9 (Cas9) nuclease or CRISPR/Cas9. These novel technologies have been applied to efficiently and specifically modify genetic information in target and effector cells. In particular, CRISPR/Cas9 technology has been applied to various hematologic malignancies and has also been used to modify and improve chimeric antigen receptor-modified T cells for the purpose of providing effective cellular therapies. Although gene editing is in its infancy in malignant hematologic diseases, there is much room for growth and application in the future. Copyright © 2018 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Technical considerations for the use of CRISPR/Cas9 in hematology research.

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Gundry, Michael C; Dever, Daniel P; Yudovich, David; Bauer, Daniel E; Haas, Simon; Wilkinson, Adam C; Singbrant, Sofie

2017-10-01

The hematopoietic system is responsible for transporting oxygen and nutrients, fighting infections, and repairing tissue damage. Hematopoietic system dysfunction therefore causes a range of serious health consequences. Lifelong hematopoiesis is maintained by repopulating multipotent hematopoietic stem cells (HSCs) that replenish shorter-lived, mature blood cell types. A prokaryotic mechanism of immunity, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system, has been recently "repurposed" to mutate mammalian genomes efficiently and in a sequence-specific manner. The application of this genome-editing technology to hematology has afforded new approaches for functional genomics and even the prospect of "correcting" dysfunctional HSCs in the treatment of serious genetic hematological diseases. In this Perspective, we provide an overview of three recent CRISPR/Cas9 methods in hematology: gene disruption, gene targeting, and saturating mutagenesis. We also summarize the technical considerations and advice provided during the May 2017 International Society of Experimental Hematology New Investigator Committee webinar on the same topic. Copyright © 2017 ISEH – Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

The role of Cas8 in type I CRISPR interference.

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Cass, Simon D B; Haas, Karina A; Stoll, Britta; Alkhnbashi, Omer S; Sharma, Kundan; Urlaub, Henning; Backofen, Rolf; Marchfelder, Anita; Bolt, Edward L

2015-05-05

CRISPR (clustered regularly interspaced short palindromic repeat) systems provide bacteria and archaea with adaptive immunity to repel invasive genetic elements. Type I systems use 'cascade' [CRISPR-associated (Cas) complex for antiviral defence] ribonucleoprotein complexes to target invader DNA, by base pairing CRISPR RNA (crRNA) to protospacers. Cascade identifies PAMs (protospacer adjacent motifs) on invader DNA, triggering R-loop formation and subsequent DNA degradation by Cas3. Cas8 is a candidate PAM recognition factor in some cascades. We analysed Cas8 homologues from type IB CRISPR systems in archaea Haloferax volcanii (Hvo) and Methanothermobacter thermautotrophicus (Mth). Cas8 was essential for CRISPR interference in Hvo and purified Mth Cas8 protein responded to PAM sequence when binding to nucleic acids. Cas8 interacted physically with Cas5-Cas7-crRNA complex, stimulating binding to PAM containing substrates. Mutation of conserved Cas8 amino acid residues abolished interference in vivo and altered catalytic activity of Cas8 protein in vitro. This is experimental evidence that Cas8 is important for targeting Cascade to invader DNA. © 2015 Authors.

[Advances in molecular mechanisms of adaptive immunity mediated by type I-E CRISPR/Cas system--A review].

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Sun, Dongchang; Qiu, Juanping

2016-01-04

To better adapt to the environment, prokaryocyte can take up exogenous genes (from bacteriophages, plasmids or genomes of other species) through horizontal gene transfer. Accompanied by the acquisition of exogenous genes, prokaryocyte is challenged by the invasion of 'selfish genes'. Therefore, to protect against the risk of gene transfer, prokaryocyte needs to establish mechanisms for selectively taking up or degrading exogenous DNA. In recent years, researchers discovered an adaptive immunity, which is mediated by the small RNA guided DNA degradation, prevents the invasion of exogenous genes in prokaryocyte. During the immune process, partial DNA fragments are firstly integrated.to the clustered regularly interspaced short palindromic repeats (CRISPR) located within the genome DNA, and then the mature CRISPR RNA transcript and the CRISPR associated proteins (Cas) form a complex CRISPR/Cas for degrading exogenous DNA. In this review, we will first briefly describe the CRISPR/Cas systems and then mainly focus on the recent advances of the function mechanism and the regulation mechanism of the type I-E CRISPR/Cas system in Escherichia coli.

Somatic Cell Nuclear Transfer Followed by CRIPSR/Cas9 Microinjection Results in Highly Efficient Genome Editing in Cloned Pigs

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Timothy P. Sheets

2016-12-01

Full Text Available The domestic pig is an ideal “dual purpose” animal model for agricultural and biomedical research. With the availability of genome editing tools such as clustered regularly interspaced short palindromic repeat (CRISPR and associated nuclease Cas9 (CRISPR/Cas9, it is now possible to perform site-specific alterations with relative ease, and will likely help realize the potential of this valuable model. In this article, we investigated for the first time a combination of somatic cell nuclear transfer (SCNT and direct injection of CRISPR/Cas ribonucleoprotein complex targeting GRB10 into the reconstituted oocytes to generate GRB10 ablated Ossabaw fetuses. This strategy resulted in highly efficient (100% generation of biallelic modifications in cloned fetuses. By combining SCNT with CRISPR/Cas9 microinjection, genome edited animals can now be produced without the need to manage a founder herd, while simultaneously eliminating the need for laborious in vitro culture and screening. Our approach utilizes standard cloning techniques while simultaneously performing genome editing in the cloned zygotes of a large animal model for agriculture and biomedical applications.

Applications of CRISPR/Cas System to Bacterial Metabolic Engineering

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Suhyung Cho

2018-04-01

Full Text Available The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas adaptive immune system has been extensively used for gene editing, including gene deletion, insertion, and replacement in bacterial and eukaryotic cells owing to its simple, rapid, and efficient activities in unprecedented resolution. Furthermore, the CRISPR interference (CRISPRi system including deactivated Cas9 (dCas9 with inactivated endonuclease activity has been further investigated for regulation of the target gene transiently or constitutively, avoiding cell death by disruption of genome. This review discusses the applications of CRISPR/Cas for genome editing in various bacterial systems and their applications. In particular, CRISPR technology has been used for the production of metabolites of high industrial significance, including biochemical, biofuel, and pharmaceutical products/precursors in bacteria. Here, we focus on methods to increase the productivity and yield/titer scan by controlling metabolic flux through individual or combinatorial use of CRISPR/Cas and CRISPRi systems with introduction of synthetic pathway in industrially common bacteria including Escherichia coli. Further, we discuss additional useful applications of the CRISPR/Cas system, including its use in functional genomics.

Cas9 specifies functional viral targets during CRISPR-Cas adaptation.

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Heler, Robert; Samai, Poulami; Modell, Joshua W; Weiner, Catherine; Goldberg, Gregory W; Bikard, David; Marraffini, Luciano A

2015-03-12

Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral infection in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets (protospacers). Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) sequence immediately downstream of the viral target. It is not known whether and how viral sequences flanked by the correct PAM are chosen as new spacers. Here we show that Cas9 selects functional spacers by recognizing their PAM during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. These results establish a new function for Cas9 in the genesis of prokaryotic immunological memory.

Disruption of HPV16-E7 by CRISPR/Cas System Induces Apoptosis and Growth Inhibition in HPV16 Positive Human Cervical Cancer Cells

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Zheng Hu

2014-01-01

Full Text Available High-risk human papillomavirus (HR-HPV has been recognized as a major causative agent for cervical cancer. Upon HPV infection, early genes E6 and E7 play important roles in maintaining malignant phenotype of cervical cancer cells. By using clustered regularly interspaced short palindromic repeats- (CRISPR- associated protein system (CRISPR/Cas system, a widely used genome editing tool in many organisms, to target HPV16-E7 DNA in HPV positive cell lines, we showed for the first time that the HPV16-E7 single-guide RNA (sgRNA guided CRISPR/Cas system could disrupt HPV16-E7 DNA at specific sites, inducing apoptosis and growth inhibition in HPV positive SiHa and Caski cells, but not in HPV negative C33A and HEK293 cells. Moreover, disruption of E7 DNA directly leads to downregulation of E7 protein and upregulation of tumor suppressor protein pRb. Therefore, our results suggest that HPV16-E7 gRNA guided CRISPR/Cas system might be used as a therapeutic strategy for the treatment of cervical cancer.

Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system.

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Liang, Zhen; Zhang, Kang; Chen, Kunling; Gao, Caixia

2014-02-20

Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have emerged as powerful tools for genome editing in a variety of species. Here, we report, for the first time, targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. We designed five TALENs targeting 4 genes, namely ZmPDS, ZmIPK1A, ZmIPK, ZmMRP4, and obtained targeting efficiencies of up to 23.1% in protoplasts, and about 13.3% to 39.1% of the transgenic plants were somatic mutations. Also, we constructed two gRNAs targeting the ZmIPK gene in maize protoplasts, at frequencies of 16.4% and 19.1%, respectively. In addition, the CRISPR/Cas system induced targeted mutations in Z. mays protoplasts with efficiencies (13.1%) similar to those obtained with TALENs (9.1%). Our results show that both TALENs and the CRISPR/Cas system can be used for genome modification in maize. Copyright © 2013. Published by Elsevier Ltd.

Function of the CRISPR-Cas System of the Human Pathogen Clostridium difficile

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Boudry, Pierre; Semenova, Ekaterina; Monot, Marc; Datsenko, Kirill A.; Lopatina, Anna; Sekulovic, Ognjen; Ospina-Bedoya, Maicol; Fortier, Louis-Charles; Severinov, Konstantin; Dupuy, Bruno

2015-01-01

ABSTRACT Clostridium difficile is the cause of most frequently occurring nosocomial diarrhea worldwide. As an enteropathogen, C. difficile must be exposed to multiple exogenous genetic elements in bacteriophage-rich gut communities. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems allow bacteria to adapt to foreign genetic invaders. Our recent data revealed active expression and processing of CRISPR RNAs from multiple type I-B CRISPR arrays in C. difficile reference strain 630. Here, we demonstrate active expression of CRISPR arrays in strain R20291, an epidemic C. difficile strain. Through genome sequencing and host range analysis of several new C. difficile phages and plasmid conjugation experiments, we provide evidence of defensive function of the CRISPR-Cas system in both C. difficile strains. We further demonstrate that C. difficile Cas proteins are capable of interference in a heterologous host, Escherichia coli. These data set the stage for mechanistic and physiological analyses of CRISPR-Cas-mediated interactions of important global human pathogen with its genetic parasites. PMID:26330515

Safety, Security, and Policy Considerations for Plant Genome Editing.

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Wolt, Jeffrey D

2017-01-01

Genome editing with engineered nucleases (GEEN) is increasingly used as a tool for gene discovery and trait development in crops through generation of targeted changes in endogenous genes. The development of the CRISPR-Cas9 system (clustered regularly interspaced short palindromic repeats with associated Cas9 protein), in particular, has enabled widespread use of genome editing. Research to date has not comprehensively addressed genome-editing specificity and off-target mismatches that may result in unintended changes within plant genomes or the potential for gene drive initiation. Governance and regulatory considerations for bioengineered crops derived from using GEEN will require greater clarity as to target specificity, the potential for mismatched edits, unanticipated downstream effects of off-target mutations, and assurance that genome reagents do not occur in finished products. Since governance and regulatory decision making involves robust standards of evidence extending from the laboratory to the postcommercial marketplace, developers of genome-edited crops must anticipate significant engagement and investment to address questions of regulators and civil society. © 2017 Elsevier Inc. All rights reserved.

CRISPR Mediated Genome Engineering and its Application in Industry.

Science.gov (United States)

Kaboli, Saeed; Babazada, Hasan

2018-01-01

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) method has been dramatically changing the field of genome engineering. It is a rapid, highly efficient and versatile tool for precise modification of genome that uses a guide RNA (gRNA) to target Cas9 to a specific sequence. This novel RNA-guided genome-editing technique has become a revolutionary tool in biomedical science and has many innovative applications in different fields. In this review, we briefly introduce the Cas9-mediated genome-editing tool, summarize the recent advances in CRISPR/Cas9 technology to engineer the genomes of a wide variety of organisms, and discuss their applications to treatment of fungal and viral disease. We also discuss advantageous of CRISPR/Cas9 technology to drug design, creation of animal model, and to food, agricultural and energy sciences. Adoption of the CRISPR/Cas9 technology in biomedical and biotechnological researches would create innovative applications of it not only for breeding of strains exhibiting desired traits for specific industrial and medical applications, but also for investigation of genome function.

Engineering synthetic TALE and CRISPR/Cas9 transcription factors for regulating gene expression.

Science.gov (United States)

Kabadi, Ami M; Gersbach, Charles A

2014-09-01

Engineered DNA-binding proteins that can be targeted to specific sites in the genome to manipulate gene expression have enabled many advances in biomedical research. This includes generating tools to study fundamental aspects of gene regulation and the development of a new class of gene therapies that alter the expression of endogenous genes. Designed transcription factors have entered clinical trials for the treatment of human diseases and others are in preclinical development. High-throughput and user-friendly platforms for designing synthetic DNA-binding proteins present innovative methods for deciphering cell biology and designing custom synthetic gene circuits. We review two platforms for designing synthetic transcription factors for manipulating gene expression: Transcription activator-like effectors (TALEs) and the RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. We present an overview of each technology and a guide for designing and assembling custom TALE- and CRISPR/Cas9-based transcription factors. We also discuss characteristics of each platform that are best suited for different applications. Copyright © 2014 Elsevier Inc. All rights reserved.

High-efficiency targeted editing of large viral genomes by RNA-guided nucleases.

Science.gov (United States)

Bi, Yanwei; Sun, Le; Gao, Dandan; Ding, Chen; Li, Zhihua; Li, Yadong; Cun, Wei; Li, Qihan

2014-05-01

A facile and efficient method for the precise editing of large viral genomes is required for the selection of attenuated vaccine strains and the construction of gene therapy vectors. The type II prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)) RNA-guided nuclease system can be introduced into host cells during viral replication. The CRISPR-Cas9 system robustly stimulates targeted double-stranded breaks in the genomes of DNA viruses, where the non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways can be exploited to introduce site-specific indels or insert heterologous genes with high frequency. Furthermore, CRISPR-Cas9 can specifically inhibit the replication of the original virus, thereby significantly increasing the abundance of the recombinant virus among progeny virus. As a result, purified recombinant virus can be obtained with only a single round of selection. In this study, we used recombinant adenovirus and type I herpes simplex virus as examples to demonstrate that the CRISPR-Cas9 system is a valuable tool for editing the genomes of large DNA viruses.

High-efficiency targeted editing of large viral genomes by RNA-guided nucleases.

Directory of Open Access Journals (Sweden)

Yanwei Bi

2014-05-01

Full Text Available A facile and efficient method for the precise editing of large viral genomes is required for the selection of attenuated vaccine strains and the construction of gene therapy vectors. The type II prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR-associated (Cas RNA-guided nuclease system can be introduced into host cells during viral replication. The CRISPR-Cas9 system robustly stimulates targeted double-stranded breaks in the genomes of DNA viruses, where the non-homologous end joining (NHEJ and homology-directed repair (HDR pathways can be exploited to introduce site-specific indels or insert heterologous genes with high frequency. Furthermore, CRISPR-Cas9 can specifically inhibit the replication of the original virus, thereby significantly increasing the abundance of the recombinant virus among progeny virus. As a result, purified recombinant virus can be obtained with only a single round of selection. In this study, we used recombinant adenovirus and type I herpes simplex virus as examples to demonstrate that the CRISPR-Cas9 system is a valuable tool for editing the genomes of large DNA viruses.

Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies

Science.gov (United States)

Richardson, Ruth E.; Suzuki, Yo

2015-01-01

Numerous DNA assembly technologies exist for generating plasmids for biological studies. Many procedures require complex in vitro or in vivo assembly reactions followed by plasmid propagation in recombination-impaired Escherichia coli strains such as DH5α, which are optimal for stable amplification of the DNA materials. Here we show that despite its utility as a cloning strain, DH5α retains sufficient recombinase activity to assemble up to six double-stranded DNA fragments ranging in size from 150 bp to at least 7 kb into plasmids in vivo. This process also requires surprisingly small amounts of DNA, potentially obviating the need for upstream assembly processes associated with most common applications of DNA assembly. We demonstrate the application of this process in cloning of various DNA fragments including synthetic genes, preparation of knockout constructs, and incorporation of guide RNA sequences in constructs for clustered regularly interspaced short palindromic repeats (CRISPR) genome editing. This consolidated process for assembly and amplification in a widely available strain of E. coli may enable productivity gain across disciplines involving recombinant DNA work. PMID:26348330

Cas5d Protein Processes Pre-crRNA and Assembles into a Cascade-like Interference Complex in Subtype I-C/Dvulg CRISPR-Cas System

Energy Technology Data Exchange (ETDEWEB)

Nam, Ki Hyun; Haitjema, Charles; Liu, Xueqi; Ding, Fran; Wang, Hongwei; DeLisa, Matthew P.; Ke, Ailong (Yale); (Cornell); (Tsinghua)

2012-10-10

Clustered regularly interspaced short palindromic repeats (CRISPRs), together with an operon of CRISPR-associated (Cas) proteins, form an RNA-based prokaryotic immune system against exogenous genetic elements. Cas5 family proteins are found in several type I CRISPR-Cas systems. Here, we report the molecular function of subtype I-C/Dvulg Cas5d from Bacillus halodurans. We show that Cas5d cleaves pre-crRNA into unit length by recognizing both the hairpin structure and the 3 single stranded sequence in the CRISPR repeat region. Cas5d structure reveals a ferredoxin domain-based architecture and a catalytic triad formed by Y46, K116, and H117 residues. We further show that after pre-crRNA processing, Cas5d assembles with crRNA, Csd1, and Csd2 proteins to form a multi-sub-unit interference complex similar to Escherichia coli Cascade (CRISPR-associated complex for antiviral defense) in architecture. Our results suggest that formation of a crRNA-presenting Cascade-like complex is likely a common theme among type I CRISPR subtypes.

Genetic engineering of a temperate phage-based delivery system for CRISPR/Cas9 antimicrobials against Staphylococcus aureus.

Science.gov (United States)

Park, Joo Youn; Moon, Bo Youn; Park, Juw Won; Thornton, Justin A; Park, Yong Ho; Seo, Keun Seok

2017-03-21

Discovery of clustered, regularly interspaced, short palindromic repeats and the Cas9 RNA-guided nuclease (CRISPR/Cas9) system provides a new opportunity to create programmable gene-specific antimicrobials that are far less likely to drive resistance than conventional antibiotics. However, the practical therapeutic use of CRISPR/Cas9 is still questionable due to current shortcomings in phage-based delivery systems such as inefficient delivery, narrow host range, and potential transfer of virulence genes by generalized transduction. In this study, we demonstrate genetic engineering strategies to overcome these shortcomings by integrating CRISPR/Cas9 system into a temperate phage genome, removing major virulence genes from the host chromosome, and expanding host specificity of the phage by complementing tail fiber protein. This significantly improved the efficacy and safety of CRISPR/Cas9 antimicrobials to therapeutic levels in both in vitro and in vivo assays. The genetic engineering tools and resources established in this study are expected to provide an efficacious and safe CRISPR/Cas9 antimicrobial, broadly applicable to Staphylococcus aureus.

Comparative genomic analysis of Lactobacillus plantarum ZJ316 reveals its genetic adaptation and potential probiotic profiles.

Science.gov (United States)

Li, Ping; Li, Xuan; Gu, Qing; Lou, Xiu-Yu; Zhang, Xiao-Mei; Song, Da-Feng; Zhang, Chen

2016-08-01

In previous studies, Lactobacillus plantarum ZJ316 showed probiotic properties, such as antimicrobial activity against various pathogens and the capacity to significantly improve pig growth and pork quality. The purpose of this study was to reveal the genes potentially related to its genetic adaptation and probiotic profiles based on comparative genomic analysis. The genome sequence of L. plantarum ZJ316 was compared with those of eight L. plantarum strains deposited in GenBank. BLASTN, Mauve, and MUMmer programs were used for genome alignment and comparison. CRISPRFinder was applied for searching the clustered regularly interspaced short palindromic repeats (CRISPRs). We identified genes that encode proteins related to genetic adaptation and probiotic profiles, including carbohydrate transport and metabolism, proteolytic enzyme systems and amino acid biosynthesis, CRISPR adaptive immunity, stress responses, bile salt resistance, ability to adhere to the host intestinal wall, exopolysaccharide (EPS) biosynthesis, and bacteriocin biosynthesis. Comparative characterization of the L. plantarum ZJ316 genome provided the genetic basis for further elucidating the functional mechanisms of its probiotic properties. ZJ316 could be considered a potential probiotic candidate.

Sensitizing pathogens to antibiotics using the CRISPR-Cas system.

Science.gov (United States)

Goren, Moran; Yosef, Ido; Qimron, Udi

2017-01-01

The extensive use of antibiotics over the last century has resulted in a significant artificial selection pressure for antibiotic-resistant pathogens to evolve. Various strategies to fight these pathogens have been introduced including new antibiotics, naturally-derived enzymes/peptides that specifically target pathogens and bacteriophages that lyse these pathogens. A new tool has recently been introduced in the fight against drug-resistant pathogens-the prokaryotic defense mechanism-clustered regularly interspaced short palindromic repeats-CRISPR associated (CRISPR-Cas) system. The CRISPR-Cas system acts as a nuclease that can be guided to cleave any target DNA, allowing sophisticated, yet feasible, manipulations of pathogens. Here, we review pioneering studies that use the CRISPR-Cas system to specifically edit bacterial populations, eliminate their resistance genes and combine these two strategies in order to produce an artificial selection pressure for antibiotic-sensitive pathogens. We suggest that intelligent design of this system, along with efficient delivery tools into pathogens, may significantly reduce the threat of antibiotic-resistant pathogens. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparative genomic analysis of Lactobacillus plantarum ZJ316 reveals its genetic adaptation and potential probiotic profiles* #

Science.gov (United States)

Li, Ping; Li, Xuan; Gu, Qing; Lou, Xiu-yu; Zhang, Xiao-mei; Song, Da-feng; Zhang, Chen

2016-01-01

Objective: In previous studies, Lactobacillus plantarum ZJ316 showed probiotic properties, such as antimicrobial activity against various pathogens and the capacity to significantly improve pig growth and pork quality. The purpose of this study was to reveal the genes potentially related to its genetic adaptation and probiotic profiles based on comparative genomic analysis. Methods: The genome sequence of L. plantarum ZJ316 was compared with those of eight L. plantarum strains deposited in GenBank. BLASTN, Mauve, and MUMmer programs were used for genome alignment and comparison. CRISPRFinder was applied for searching the clustered regularly interspaced short palindromic repeats (CRISPRs). Results: We identified genes that encode proteins related to genetic adaptation and probiotic profiles, including carbohydrate transport and metabolism, proteolytic enzyme systems and amino acid biosynthesis, CRISPR adaptive immunity, stress responses, bile salt resistance, ability to adhere to the host intestinal wall, exopolysaccharide (EPS) biosynthesis, and bacteriocin biosynthesis. Conclusions: Comparative characterization of the L. plantarum ZJ316 genome provided the genetic basis for further elucidating the functional mechanisms of its probiotic properties. ZJ316 could be considered a potential probiotic candidate. PMID:27487802

Building Cre Knockin Rat Lines Using CRISPR/Cas9.

Science.gov (United States)

Ma, Yuanwu; Zhang, Lianfeng; Huang, Xingxu

2017-01-01

Conditional gene inactivation strategy helps researchers to study the gene functions that are critical in embryogenesis or in defined tissues of adulthood. The Cre/loxP system is widely used for conditional gene inactivation/activation in cells or organisms. Cre knockin animal lines are essential for gene expression or inactivation in a spatially and temporally restricted manner. However, to generate a Cre knockin line by traditional approach is laborious. Recently, the clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) has been proven as a simple and efficient genome-editing tool. We have used CRISPR/Cas9 system to generate rat strains that carry Cre genes in different targeted gene loci by direct delivery of gRNAs/Cas9/donors into fertilized eggs. Here, we described a stepwise procedure for the generation of Cre knockin rat, including target site selection, RNA preparation, the construction of the template donor, pronuclear injection, and the genotyping of precise Cre insertion in F 0 rats. Taken together, the establishment of Cre knockin line can be achieved within 6 weeks.

Oncogenic Human Papillomavirus: Application of CRISPR/Cas9 Therapeutic Strategies for Cervical Cancer

Directory of Open Access Journals (Sweden)

Shuai Zhen

2017-12-01

Full Text Available Oncogenic human papillomaviruses (HPVs cause different types of cancer especially cervical cancer. HPV-associated carcinogenesis provides a classical model system for clustered regularly interspaced short palindromic repeats (CRISPR/Cas9 based cancer therapies since the viral oncogenes E6 and E7 are exclusively expressed in cancerous cells. Sequence-specific gene knockdown/knockout using CRISPR/Cas9 shows promise as a novel therapeutic approach for the treatment of a variety of diseases that currently lack effective treatments. However, CRISPR/Cas9-based targeting therapy requires further validation of its efficacy in vitro and in vivo to eliminate the potential off-target effects, necessitates verification of the delivery vehicles and the combinatory use of conventional therapies with CRISPR/Cas9 to ensure the feasibility and safety. In this review we discuss the potential of combining CRISPR/Cas9 with other treatment options as therapies for oncogenic HPVs-associated carcinogenesis. and present our assessment of the promising path to the development of CRISPR/Cas9 therapeutic strategies for clinical settings.

Mutations in the Plasmodium falciparum Cyclic Amine Resistance Locus (PfCARL Confer Multidrug Resistance

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Gregory LaMonte

2016-07-01

Full Text Available Mutations in the Plasmodium falciparum cyclic amine resistance locus (PfCARL are associated with parasite resistance to the imidazolopiperazines, a potent class of novel antimalarial compounds that display both prophylactic and transmission-blocking activity, in addition to activity against blood-stage parasites. Here, we show that pfcarl encodes a protein, with a predicted molecular weight of 153 kDa, that localizes to the cis-Golgi apparatus of the parasite in both asexual and sexual blood stages. Utilizing clustered regularly interspaced short palindromic repeat (CRISPR-mediated gene introduction of 5 variants (L830V, S1076N/I, V1103L, and I1139K, we demonstrate that mutations in pfcarl are sufficient to generate resistance against the imidazolopiperazines in both asexual and sexual blood-stage parasites. We further determined that the mutant PfCARL protein confers resistance to several structurally unrelated compounds. These data suggest that PfCARL modulates the levels of small-molecule inhibitors that affect Golgi-related processes, such as protein sorting or membrane trafficking, and is therefore an important mechanism of resistance in malaria parasites.

Structure of a CRISPR-associated protein Cas2 from Desulfovibrio vulgaris

Energy Technology Data Exchange (ETDEWEB)

Samai, Poulami; Smith, Paul; Shuman, Stewart [Molecular Biology Program, Sloan-Kettering Institute for Cancer Research (United States)

2010-12-01

A 1.35 Å resolution crystal structure of Cas2 from the bacterium Desulfovibrio vulgaris (DvuCas2) is reported. CRISPRs (clustered regularly interspaced short palindromic repeats) provide bacteria and archaea with RNA-guided acquired immunity to invasive DNAs. CRISPR-associated (Cas) proteins carry out the immune effector functions. Cas2 is a universal component of the CRISPR system. Here, a 1.35 Å resolution crystal structure of Cas2 from the bacterium Desulfovibrio vulgaris (DvuCas2) is reported. DvuCas2 is a homodimer, with each protomer consisting of an N-terminal βαββαβ ferredoxin fold (amino acids 1–78) to which is appended a C-terminal segment (amino acids 79–102) that includes a short 3{sub 10}-helix and a fifth β-strand. The β5 strands align with the β4 strands of the opposite protomers, resulting in two five-stranded antiparallel β-sheets that form a sandwich at the dimer interface. The DvuCas2 dimer is stabilized by a distinctive network of hydrophilic cross-protomer side-chain interactions.

Structure of a CRISPR-associated protein Cas2 from Desulfovibrio vulgaris

International Nuclear Information System (INIS)

Samai, Poulami; Smith, Paul; Shuman, Stewart

2010-01-01

A 1.35 Å resolution crystal structure of Cas2 from the bacterium Desulfovibrio vulgaris (DvuCas2) is reported. CRISPRs (clustered regularly interspaced short palindromic repeats) provide bacteria and archaea with RNA-guided acquired immunity to invasive DNAs. CRISPR-associated (Cas) proteins carry out the immune effector functions. Cas2 is a universal component of the CRISPR system. Here, a 1.35 Å resolution crystal structure of Cas2 from the bacterium Desulfovibrio vulgaris (DvuCas2) is reported. DvuCas2 is a homodimer, with each protomer consisting of an N-terminal βαββαβ ferredoxin fold (amino acids 1–78) to which is appended a C-terminal segment (amino acids 79–102) that includes a short 3 10 -helix and a fifth β-strand. The β5 strands align with the β4 strands of the opposite protomers, resulting in two five-stranded antiparallel β-sheets that form a sandwich at the dimer interface. The DvuCas2 dimer is stabilized by a distinctive network of hydrophilic cross-protomer side-chain interactions

Metabolic engineering tools in model cyanobacteria.

Science.gov (United States)

Carroll, Austin L; Case, Anna E; Zhang, Angela; Atsumi, Shota

2018-03-26

Developing sustainable routes for producing chemicals and fuels is one of the most important challenges in metabolic engineering. Photoautotrophic hosts are particularly attractive because of their potential to utilize light as an energy source and CO 2 as a carbon substrate through photosynthesis. Cyanobacteria are unicellular organisms capable of photosynthesis and CO 2 fixation. While engineering in heterotrophs, such as Escherichia coli, has result in a plethora of tools for strain development and hosts capable of producing valuable chemicals efficiently, these techniques are not always directly transferable to cyanobacteria. However, recent efforts have led to an increase in the scope and scale of chemicals that cyanobacteria can produce. Adaptations of important metabolic engineering tools have also been optimized to function in photoautotrophic hosts, which include Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, 13 C Metabolic Flux Analysis (MFA), and Genome-Scale Modeling (GSM). This review explores innovations in cyanobacterial metabolic engineering, and highlights how photoautotrophic metabolism has shaped their development. Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Benchmarking CRISPR on-target sgRNA design.

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Yan, Jifang; Chuai, Guohui; Zhou, Chi; Zhu, Chenyu; Yang, Jing; Zhang, Chao; Gu, Feng; Xu, Han; Wei, Jia; Liu, Qi

2017-02-15

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based gene editing has been widely implemented in various cell types and organisms. A major challenge in the effective application of the CRISPR system is the need to design highly efficient single-guide RNA (sgRNA) with minimal off-target cleavage. Several tools are available for sgRNA design, while limited tools were compared. In our opinion, benchmarking the performance of the available tools and indicating their applicable scenarios are important issues. Moreover, whether the reported sgRNA design rules are reproducible across different sgRNA libraries, cell types and organisms remains unclear. In our study, a systematic and unbiased benchmark of the sgRNA predicting efficacy was performed on nine representative on-target design tools, based on six benchmark data sets covering five different cell types. The benchmark study presented here provides novel quantitative insights into the available CRISPR tools. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Activity and specificity of TRV-mediated gene editing in plants

KAUST Repository

Ali, Zahir

2015-06-03

© 2015 Taylor and Francis Group, LLC. Plant trait engineering requires efficient targeted genome-editing technologies. Clustered regularly interspaced palindromic repeats (CRISPRs)/ CRISPR associated (Cas) type II system is used for targeted genome-editing applications across eukaryotic species including plants. Delivery of genome engineering reagents and recovery of mutants remain challenging tasks for in planta applications. Recently, we reported the development of Tobacco rattle virus (TRV)-mediated genome editing in Nicotiana benthamiana. TRV infects the growing points and possesses small genome size; which facilitate cloning, multiplexing, and agroinfections. Here, we report on the persistent activity and specificity of the TRV-mediated CRISPR/Cas9 system for targeted modification of the Nicotiana benthamiana genome. Our data reveal the persistence of the TRVmediated Cas9 activity for up to 30 d post-agroinefection. Further, our data indicate that TRV-mediated genome editing exhibited no off-target activities at potential off-targets indicating the precision of the system for plant genome engineering. Taken together, our data establish the feasibility and exciting possibilities of using virus-mediated CRISPR/Cas9 for targeted engineering of plant genomes.

Singular continuous spectrum for palindromic Schroedinger operators

International Nuclear Information System (INIS)

Hof, A.; Knill, O.; Simon, B.

1995-01-01

We give new examples of discrete Schroedinger operators with potentials taking finitely many values that have purely singular continuous spectrum. If the hull X of the potential is strictly ergodic, then the existence of just one potential x in X for which the operator has no eigenvalues implies that there is a generic set in X for which the operator has purely singular continuous spectrum. A sufficient condition for the existence of such an x is that there is a z element of X that contains arbitrarily long palindromes. Thus we can define a large class of primitive substitutions for which the operators are purely singularly continuous for a generic subset in X. The class includes well-known substitutions like Fibonacci, Thue-Morse, Period Doubling, binary non-Pisot and ternary non-Pisot. We also show that the operator has no absolutely continuous spectrum for all x element of X if X derives from a primitive substitution. For potentials defined by circle maps, x n =l J (θ 0 +nα), we show that the operator has purely singular continuous spectrum for a generic subset in X for all irrational α and every half-open interval J. (orig.)

CRISPR/Cas9 advances engineering of microbial cell factories

DEFF Research Database (Denmark)

Jakociunas, Tadas; Jensen, Michael Krogh; Keasling, Jay D.

2016-01-01

interspaced palindromic repeats (CRISPR) and its associated proteins (Cas) have become the method of choice for precision genome engineering in many organisms due to their orthogonality, versatility and efficacy. Here we review the strategies adopted for implementation of RNA-guided CRISPR/Cas9 genome editing......-RNAs will be highlighted. Finally, this review will provide a perspective on the immediate challenges and opportunities foreseen by the use of CRISPR/Cas9 genome engineering and regulation in the context of metabolic engineering....

Distribution of antimicrobial resistance determinants, virulence-associated factors and clustered regularly interspaced palindromic repeats loci in isolates of Enterococcus faecalis from various settings and genetic lineages.

Science.gov (United States)

Gawryszewska, Iwona; Malinowska, Katarzyna; Kuch, Alicja; Chrobak-Chmiel, Dorota; Trokenheim, Lucja Laniewska-; Hryniewicz, Waleria; Sadowy, Ewa

2017-03-01

Enterococcus faecalis represents an important factor of hospital-associated infections (HAIs). The knowledge on its evolution from a commensal to an opportunistic pathogen is still limited; thus, we performed a study to characterise distribution of factors that may contribute to this adaptation. Using a collection obtained from various settings (hospitalised patients, community carriers, animals, fresh food, sewage, water), we investigated differences in antimicrobial susceptibility, distribution of antimicrobial resistance genes, virulence-associated determinants and phenotypes, and CRISPR loci in the context of the clonal relatedness of isolates. Bayesian Analysis of Population Structure revealed the presence of three major groups; two subgroups comprised almost exclusively HAI isolates, belonging to previously proposed enterococcal high-risk clonal complexes (HiRECCs) 6 and 28. Isolates of these two subgroups were significantly enriched in antimicrobial resistance genes, presumably produced a polysaccharide capsule and often carried the aggregation substance asa1; distribution of other virulence-associated genes, such as esp and cyl, formation of a biofilm and gelatinase production were more variable. Moreover, both subgroups showed a low prevalence of CRISPR-Cas 1 and 3 and presence of small CRISPR2 variants. Our study confirms the importance of HiRECCs in the population of E. faecalis and their confinement to the hospital settings. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Analysis of the type II-A CRISPR-Cas system of Streptococcus agalactiae reveals distinctive features according to genetic lineages

Science.gov (United States)

Lier, Clément; Baticle, Elodie; Horvath, Philippe; Haguenoer, Eve; Valentin, Anne-Sophie; Glaser, Philippe; Mereghetti, Laurent; Lanotte, Philippe

2015-01-01

CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) are found in 90% of archaea and about 40% of bacteria. In this original system, CRISPR arrays comprise short, almost unique sequences called spacers that are interspersed with conserved palindromic repeats. These systems play a role in adaptive immunity and participate to fight non-self DNA such as integrative and conjugative elements, plasmids, and phages. In Streptococcus agalactiae, a bacterium implicated in colonization and infections in humans since the 1960s, two CRISPR-Cas systems have been described. A type II-A system, characterized by proteins Cas9, Cas1, Cas2, and Csn2, is ubiquitous, and a type I–C system, with the Cas8c signature protein, is present in about 20% of the isolates. Unlike type I–C, which appears to be non-functional, type II-A appears fully functional. Here we studied type II-A CRISPR-cas loci from 126 human isolates of S. agalactiae belonging to different clonal complexes that represent the diversity of the species and that have been implicated in colonization or infection. The CRISPR-cas locus was analyzed both at spacer and repeat levels. Major distinctive features were identified according to the phylogenetic lineages previously defined by multilocus sequence typing, especially for the sequence type (ST) 17, which is considered hypervirulent. Among other idiosyncrasies, ST-17 shows a significantly lower number of spacers in comparison with other lineages. This characteristic could reflect the peculiar virulence or colonization specificities of this lineage. PMID:26124774

Modelling the diurnal and seasonal dynamics of soil CO2 exchange in a semiarid ecosystem with high plant-interspace heterogeneity

Science.gov (United States)

Gong, Jinnan; Wang, Ben; Jia, Xin; Feng, Wei; Zha, Tianshan; Kellomäki, Seppo; Peltola, Heli

2018-01-01

We used process-based modelling to investigate the roles of carbon-flux (C-flux) components and plant-interspace heterogeneities in regulating soil CO2 exchanges (FS) in a dryland ecosystem with sparse vegetation. To simulate the diurnal and seasonal dynamics of FS, the modelling considered simultaneously the CO2 production, transport and surface exchanges (e.g. biocrust photosynthesis, respiration and photodegradation). The model was parameterized and validated with multivariate data measured during the years 2013-2014 in a semiarid shrubland ecosystem in Yanchi, northwestern China. The model simulation showed that soil rewetting could enhance CO2 dissolution and delay the emission of CO2 produced from rooting zone. In addition, an ineligible fraction of respired CO2 might be removed from soil volumes under respiration chambers by lateral water flows and root uptakes. During rewetting, the lichen-crusted soil could shift temporally from net CO2 source to sink due to the activated photosynthesis of biocrust but the restricted CO2 emissions from subsoil. The presence of plant cover could decrease the root-zone CO2 production and biocrust C sequestration but increase the temperature sensitivities of these fluxes. On the other hand, the sensitivities of root-zone emissions to water content were lower under canopy, which may be due to the advection of water flows from the interspace to canopy. To conclude, the complexity and plant-interspace heterogeneities of soil C processes should be carefully considered to extrapolate findings from chamber to ecosystem scales and to predict the ecosystem responses to climate change and extreme climatic events. Our model can serve as a useful tool to simulate the soil CO2 efflux dynamics in dryland ecosystems.

UNFOLDED REGULAR AND SEMI-REGULAR POLYHEDRA

Directory of Open Access Journals (Sweden)

IONIŢĂ Elena

2015-06-01

Full Text Available This paper proposes a presentation unfolding regular and semi-regular polyhedra. Regular polyhedra are convex polyhedra whose faces are regular and equal polygons, with the same number of sides, and whose polyhedral angles are also regular and equal. Semi-regular polyhedra are convex polyhedra with regular polygon faces, several types and equal solid angles of the same type. A net of a polyhedron is a collection of edges in the plane which are the unfolded edges of the solid. Modeling and unfolding Platonic and Arhimediene polyhedra will be using 3dsMAX program. This paper is intended as an example of descriptive geometry applications.

Modelling the diurnal and seasonal dynamics of soil CO2 exchange in a semiarid ecosystem with high plant–interspace heterogeneity

Directory of Open Access Journals (Sweden)

J. Gong

2018-01-01

Full Text Available We used process-based modelling to investigate the roles of carbon-flux (C-flux components and plant–interspace heterogeneities in regulating soil CO2 exchanges (FS in a dryland ecosystem with sparse vegetation. To simulate the diurnal and seasonal dynamics of FS, the modelling considered simultaneously the CO2 production, transport and surface exchanges (e.g. biocrust photosynthesis, respiration and photodegradation. The model was parameterized and validated with multivariate data measured during the years 2013–2014 in a semiarid shrubland ecosystem in Yanchi, northwestern China. The model simulation showed that soil rewetting could enhance CO2 dissolution and delay the emission of CO2 produced from rooting zone. In addition, an ineligible fraction of respired CO2 might be removed from soil volumes under respiration chambers by lateral water flows and root uptakes. During rewetting, the lichen-crusted soil could shift temporally from net CO2 source to sink due to the activated photosynthesis of biocrust but the restricted CO2 emissions from subsoil. The presence of plant cover could decrease the root-zone CO2 production and biocrust C sequestration but increase the temperature sensitivities of these fluxes. On the other hand, the sensitivities of root-zone emissions to water content were lower under canopy, which may be due to the advection of water flows from the interspace to canopy. To conclude, the complexity and plant–interspace heterogeneities of soil C processes should be carefully considered to extrapolate findings from chamber to ecosystem scales and to predict the ecosystem responses to climate change and extreme climatic events. Our model can serve as a useful tool to simulate the soil CO2 efflux dynamics in dryland ecosystems.

Site-Specific Gene Editing of Human Hematopoietic Stem Cells for X-Linked Hyper-IgM Syndrome

Directory of Open Access Journals (Sweden)

Caroline Y. Kuo

2018-05-01

Full Text Available X-linked hyper-immunoglobulin M (hyper-IgM syndrome (XHIM is a primary immunodeficiency due to mutations in CD40 ligand that affect immunoglobulin class-switch recombination and somatic hypermutation. The disease is amenable to gene therapy using retroviral vectors, but dysregulated gene expression results in abnormal lymphoproliferation in mouse models, highlighting the need for alternative strategies. Here, we demonstrate the ability of both the transcription activator-like effector nuclease (TALEN and clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9 platforms to efficiently drive integration of a normal copy of the CD40L cDNA delivered by Adeno-Associated Virus. Site-specific insertion of the donor sequence downstream of the endogenous CD40L promoter maintained physiologic expression of CD40L while overriding all reported downstream mutations. High levels of gene modification were achieved in primary human hematopoietic stem cells (HSCs, as well as in cell lines and XHIM-patient-derived T cells. Notably, gene-corrected HSCs engrafted in immunodeficient mice at clinically relevant frequencies. These studies provide the foundation for a permanent curative therapy in XHIM.

Human Induced Pluripotent Stem Cell NEUROG2 Dual Knockin Reporter Lines Generated by the CRISPR/Cas9 System.

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Li, Shenglan; Xue, Haipeng; Wu, Jianbo; Rao, Mahendra S; Kim, Dong H; Deng, Wenbin; Liu, Ying

2015-12-15

Human induced pluripotent stem cell (hiPSC) technologies are powerful tools for modeling development and disease, drug screening, and regenerative medicine. Faithful gene targeting in hiPSCs greatly facilitates these applications. We have developed a fast and precise clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) technology-based method and obtained fluorescent protein and antibiotic resistance dual knockin reporters in hiPSC lines for neurogenin2 (NEUROG2), an important proneural transcription factor. Gene targeting efficiency was greatly improved in CRISPR/Cas9-mediated homology directed recombination (∼ 33% correctly targeted clones) compared to conventional targeting protocol (∼ 3%) at the same locus. No off-target events were detected. In addition, taking the advantage of the versatile applications of the CRISPR/Cas9 system, we designed transactivation components to transiently induce NEUROG2 expression, which helps identify transcription factor binding sites and trans-regulation regions of human NEUROG2. The strategy of using CRISPR/Cas9 genome editing coupled with fluorescence-activated cell sorting of neural progenitor cells in a knockin lineage hiPSC reporter platform might be broadly applicable in other stem cell derivatives and subpopulations.

Transcription activator-like effector nucleases mediated metabolic engineering for enhanced fatty acids production in Saccharomyces cerevisiae

KAUST Repository

Aouida, Mustapha; Li, Lixin; Mahjoub, Ali; Alshareef, Sahar; Ali, Zahir; Piatek, Agnieszka Anna; Mahfouz, Magdy M.

2015-01-01

Targeted engineering of microbial genomes holds much promise for diverse biotechnological applications. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/Cas9 systems are capable of efficiently editing microbial genomes, including that of Saccharomyces cerevisiae. Here, we demonstrate the use of TALENs to edit the genome of S.cerevisiae with the aim of inducing the overproduction of fatty acids. Heterodimeric TALENs were designed to simultaneously edit the FAA1 and FAA4 genes encoding acyl-CoA synthetases in S.cerevisiae. Functional yeast double knockouts generated using these TALENs over-produce large amounts of free fatty acids into the cell. This study demonstrates the use of TALENs for targeted engineering of yeast and demonstrates that this technology can be used to stimulate the enhanced production of free fatty acids, which are potential substrates for biofuel production. This proof-of-principle study extends the utility of TALENs as excellent genome editing tools and highlights their potential use for metabolic engineering of yeast and other organisms, such as microalgae and plants, for biofuel production. © 2015 The Society for Biotechnology, Japan.

Imipenem represses CRISPR-Cas interference of DNA acquisition through H-NS stimulation in Klebsiella pneumoniae

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Lin, Tzu-Lung; Pan, Yi-Jiun; Hsieh, Pei-Fang; Hsu, Chun-Ru; Wu, Meng-Chuan; Wang, Jin-Town

2016-01-01

Analysis of the genome of Klebsiella pneumoniae NTUH-K2044 strain revealed the presence of two clustered regularly interspaced short palindromic repeats (CRISPR) arrays separated with CRISPR-associated (cas) genes. Carbapenem-resistant K. pneumoniae isolates were observed to be less likely to have CRISPR-Cas than sensitive strains (5/85 vs. 22/132). Removal of the transcriptional repressor, H-NS, was shown to prevent the transformation of plasmids carrying a spacer and putative proto-spacer adjacent motif (PAM). The CRISPR-Cas system also decreased pUC-4K plasmid stability, resulting in plasmid loss from the bacteria with acquisition of new spacers. Analysis of the acquired proto-spacers in pUC-4K indicated that 5′-TTN-3′ was the preferred PAM in K. pneumoniae. Treatment of cells by imipenem induced hns expression, thereby decreasing cas3 expression and consequently repressed CRISPR-Cas activity resulted in increase of plasmid stability. In conclusion, NTUH-K2044 CRISPR-Cas contributes to decrease of plasmid transformation and stability. Through repression of CRISPR-Cas activity by induced H-NS, bacteria might be more able to acquire DNA to confront the challenge of imipenem. PMID:27531594

Transcription activator-like effector nucleases mediated metabolic engineering for enhanced fatty acids production in Saccharomyces cerevisiae

KAUST Repository

Aouida, Mustapha

2015-04-01

Targeted engineering of microbial genomes holds much promise for diverse biotechnological applications. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/Cas9 systems are capable of efficiently editing microbial genomes, including that of Saccharomyces cerevisiae. Here, we demonstrate the use of TALENs to edit the genome of S.cerevisiae with the aim of inducing the overproduction of fatty acids. Heterodimeric TALENs were designed to simultaneously edit the FAA1 and FAA4 genes encoding acyl-CoA synthetases in S.cerevisiae. Functional yeast double knockouts generated using these TALENs over-produce large amounts of free fatty acids into the cell. This study demonstrates the use of TALENs for targeted engineering of yeast and demonstrates that this technology can be used to stimulate the enhanced production of free fatty acids, which are potential substrates for biofuel production. This proof-of-principle study extends the utility of TALENs as excellent genome editing tools and highlights their potential use for metabolic engineering of yeast and other organisms, such as microalgae and plants, for biofuel production. © 2015 The Society for Biotechnology, Japan.

Genome engineering via TALENs and CRISPR/Cas9 systems: challenges and perspectives

KAUST Repository

Mahfouz, Magdy M.

2014-09-24

The ability to precisely modify genome sequence and regulate gene expression patterns in a site-specific manner holds much promise in plant biotechnology. Genome-engineering technologies that enable such highly specific and efficient modification are advancing with unprecedented pace. Transcription activator-like effectors (TALEs) provide customizable DNA-binding modules designed to bind to any sequence of interest. Thus, TALEs have been used as a DNA targeting module fused to functional domains for a variety of targeted genomic and epigenomic modifications. TALE nucleases (TALENs) have been used with much success across eukaryotic species to edit genomes. Recently, clustered regularly interspaced palindromic repeats (CRISPRs) that are used as guide RNAs for Cas9 nuclease-specific digestion has been introduced as a highly efficient DNA-targeting platform for genome editing and regulation. Here, we review the discovery, development and limitations of TALENs and CRIPSR/Cas9 systems as genome-engineering platforms in plants. We discuss the current questions, potential improvements and the development of the next-generation genome-editing platforms with an emphasis on producing designer plants to address the needs of agriculture and basic plant biology.

CRISPR-Cas9: a promising genetic engineering approach in cancer research

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Ratan, Zubair Ahmed; Son, Young-Jin; Uddin, Bhuiyan Mohammad Mahtab; Yusuf, Md. Abdullah; Zaman, Sojib Bin; Kim, Jong-Hoon; Banu, Laila Anjuman

2018-01-01

Bacteria and archaea possess adaptive immunity against foreign genetic materials through clustered regularly interspaced short palindromic repeat (CRISPR) systems. The discovery of this intriguing bacterial system heralded a revolutionary change in the field of medical science. The CRISPR and CRISPR-associated protein 9 (Cas9) based molecular mechanism has been applied to genome editing. This CRISPR-Cas9 technique is now able to mediate precise genetic corrections or disruptions in in vitro and in vivo environments. The accuracy and versatility of CRISPR-Cas have been capitalized upon in biological and medical research and bring new hope to cancer research. Cancer involves complex alterations and multiple mutations, translocations and chromosomal losses and gains. The ability to identify and correct such mutations is an important goal in cancer treatment. In the context of this complex cancer genomic landscape, there is a need for a simple and flexible genetic tool that can easily identify functional cancer driver genes within a comparatively short time. The CRISPR-Cas system shows promising potential for modeling, repairing and correcting genetic events in different types of cancer. This article reviews the concept of CRISPR-Cas, its application and related advantages in oncology. PMID:29434679

Rational modular design of metabolic network for efficient production of plant polyphenol pinosylvin.

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Wu, Junjun; Zhang, Xia; Zhu, Yingjie; Tan, Qinyu; He, Jiacheng; Dong, Mingsheng

2017-05-03

Efficient biosynthesis of the plant polyphenol pinosylvin, which has numerous applications in nutraceuticals and pharmaceuticals, is necessary to make biological production economically viable. To this end, an efficient Escherichia coli platform for pinosylvin production was developed via a rational modular design approach. Initially, different candidate pathway enzymes were screened to construct de novo pinosylvin pathway directly from D-glucose. A comparative analysis of pathway intermediate pools identified that this initial construct led to the intermediate cinnamic acid accumulation. The pinosylvin synthetic pathway was then divided into two new modules separated at cinnamic acid. Combinatorial optimization of transcriptional and translational levels of these two modules resulted in a 16-fold increase in pinosylvin titer. To further improve the concentration of the limiting precursor malonyl-CoA, the malonyl-CoA synthesis module based on clustered regularly interspaced short palindromic repeats interference was assembled and optimized with other two modules. The final pinosylvin titer was improved to 281 mg/L, which was the highest pinosylvin titer even directly from D-glucose without any additional precursor supplementation. The rational modular design approach described here could bolster our capabilities in synthetic biology for value-added chemical production.

MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5.

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Nezich, Catherine L; Wang, Chunxin; Fogel, Adam I; Youle, Richard J

2015-08-03

The kinase PINK1 and ubiquitin ligase Parkin can regulate the selective elimination of damaged mitochondria through autophagy (mitophagy). Because of the demand on lysosomal function by mitophagy, we investigated a role for the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, in this process. We show that during mitophagy TFEB translocates to the nucleus and displays transcriptional activity in a PINK1- and Parkin-dependent manner. MITF and TFE3, homologues of TFEB belonging to the same microphthalmia/transcription factor E (MiT/TFE) family, are similarly regulated during mitophagy. Unlike TFEB translocation after starvation-induced mammalian target of rapamycin complex 1 inhibition, Parkin-mediated TFEB relocalization required Atg9A and Atg5 activity. However, constitutively active Rag guanosine triphosphatases prevented TFEB translocation during mitophagy, suggesting cross talk between these two MiT/TFE activation pathways. Analysis of clustered regularly interspaced short palindromic repeats-generated TFEB/MITF/TFE3/TFEC single, double, and triple knockout cell lines revealed that these proteins partly facilitate Parkin-mediated mitochondrial clearance. These results illuminate a pathway leading to MiT/TFE transcription factor activation, distinct from starvation-induced autophagy, which occurs during mitophagy.

A novel multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) method for Propionibacterium acnes.

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Hauck, Yolande; Soler, Charles; Gérôme, Patrick; Vong, Rithy; Macnab, Christine; Appere, Géraldine; Vergnaud, Gilles; Pourcel, Christine

2015-07-01

Propionibacterium acnes plays a central role in the pathogenesis of acne and is responsible for severe opportunistic infections. Numerous typing schemes have been developed that allow the identification of phylotypes, but they are often insufficient to differentiate subtypes. To better understand the genetic diversity of this species and to perform epidemiological analyses, high throughput discriminant genotyping techniques are needed. Here we describe the development of a multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) method. Thirteen VNTRs were identified in the genome of P. acnes and were used to genotype a collection of clinical isolates. In addition, publically available sequencing data for 102 genomes were analyzed in silico, providing an MLVA genotype. The clustering of MLVA data was in perfect congruence with whole genome based clustering. Analysis of the clustered regularly interspaced short palindromic repeat (CRISPR) element uncovered new spacers, a supplementary source of genotypic information. The present MLVA13 scheme and associated internet database represents a first line genotyping assay to investigate large number of isolates. Particular strains may then be submitted to full genome sequencing in order to better analyze their pathogenic potential. Copyright © 2015 Elsevier B.V. All rights reserved.

Genome engineering via TALENs and CRISPR/Cas9 systems: challenges and perspectives

KAUST Repository

Mahfouz, Magdy M.; Piatek, Agnieszka Anna; Stewart, Charles Neal

2014-01-01

The ability to precisely modify genome sequence and regulate gene expression patterns in a site-specific manner holds much promise in plant biotechnology. Genome-engineering technologies that enable such highly specific and efficient modification are advancing with unprecedented pace. Transcription activator-like effectors (TALEs) provide customizable DNA-binding modules designed to bind to any sequence of interest. Thus, TALEs have been used as a DNA targeting module fused to functional domains for a variety of targeted genomic and epigenomic modifications. TALE nucleases (TALENs) have been used with much success across eukaryotic species to edit genomes. Recently, clustered regularly interspaced palindromic repeats (CRISPRs) that are used as guide RNAs for Cas9 nuclease-specific digestion has been introduced as a highly efficient DNA-targeting platform for genome editing and regulation. Here, we review the discovery, development and limitations of TALENs and CRIPSR/Cas9 systems as genome-engineering platforms in plants. We discuss the current questions, potential improvements and the development of the next-generation genome-editing platforms with an emphasis on producing designer plants to address the needs of agriculture and basic plant biology.

CRISPR/Cas9 in insects: Applications, best practices and biosafety concerns.

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Taning, Clauvis Nji Tizi; Van Eynde, Benigna; Yu, Na; Ma, Sanyuan; Smagghe, Guy

2017-04-01

Discovered as a bacterial adaptive immune system, CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeat/CRISPR associated) is being developed as an attractive tool in genome editing. Due to its high specificity and applicability, CRISPR/Cas9-mediated gene editing has been employed in a multitude of organisms and cells, including insects, for not only fundamental research such as gene function studies, but also applied research such as modification of organisms of economic importance. Despite the rapid increase in the use of CRISPR in insect genome editing, results still differ from each study, principally due to existing differences in experimental parameters, such as the Cas9 and guide RNA form, the delivery method, the target gene and off-target effects. Here, we review current reports on the successes of CRISPR/Cas9 applications in diverse insects and insect cells. We furthermore summarize several best practices to give a useful checklist of CRISPR/Cas9 experimental setup in insects for beginners. Lastly, we discuss the biosafety concerns related to the release of CRISPR/Cas9-edited insects into the environment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells.

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Dong, Zhan-Qi; Chen, Ting-Ting; Zhang, Jun; Hu, Nan; Cao, Ming-Ya; Dong, Fei-Fan; Jiang, Ya-Ming; Chen, Peng; Lu, Cheng; Pan, Min-Hui

2016-06-01

Although current antiviral strategies can inhibit baculovirus infection and decrease viral DNA replication to a certain extent, novel tools are required for specific and accurate elimination of baculovirus genomes from infected insects. Using the newly developed clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology, we disrupted a viral genome in infected insect cells in vitro as a defense against viral infection. We optimized the CRISPR/Cas9 system to edit foreign and viral genome in insect cells. Using Bombyx mori nucleopolyhedrovirus (BmNPV) as a model, we found that the CRISPR/Cas9 system was capable of cleaving the replication key factor ie-1 in BmNPV thus effectively inhibiting virus proliferation. Furthermore, we constructed a virus-inducible CRISPR/Cas9 editing system, which minimized the probability of off-target effects and was rapidly activated after viral infection. This is the first report describing the application of the CRISPR/Cas9 system in insect antiviral research. Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells provides insights to produce virus-resistant transgenic strains for future. Copyright © 2016 Elsevier B.V. All rights reserved.

CRISPR-Cas9-Edited Site Sequencing (CRES-Seq): An Efficient and High-Throughput Method for the Selection of CRISPR-Cas9-Edited Clones.

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Veeranagouda, Yaligara; Debono-Lagneaux, Delphine; Fournet, Hamida; Thill, Gilbert; Didier, Michel

2018-01-16

The emergence of clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) gene editing systems has enabled the creation of specific mutants at low cost, in a short time and with high efficiency, in eukaryotic cells. Since a CRISPR-Cas9 system typically creates an array of mutations in targeted sites, a successful gene editing project requires careful selection of edited clones. This process can be very challenging, especially when working with multiallelic genes and/or polyploid cells (such as cancer and plants cells). Here we described a next-generation sequencing method called CRISPR-Cas9 Edited Site Sequencing (CRES-Seq) for the efficient and high-throughput screening of CRISPR-Cas9-edited clones. CRES-Seq facilitates the precise genotyping up to 96 CRISPR-Cas9-edited sites (CRES) in a single MiniSeq (Illumina) run with an approximate sequencing cost of $6/clone. CRES-Seq is particularly useful when multiple genes are simultaneously targeted by CRISPR-Cas9, and also for screening of clones generated from multiallelic genes/polyploid cells. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

Advancing chimeric antigen receptor T cell therapy with CRISPR/Cas9.

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Ren, Jiangtao; Zhao, Yangbing

2017-09-01

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (CRISPR/Cas9) system, an RNA-guided DNA targeting technology, is triggering a revolution in the field of biology. CRISPR/Cas9 has demonstrated great potential for genetic manipulation. In this review, we discuss the current development of CRISPR/Cas9 technologies for therapeutic applications, especially chimeric antigen receptor (CAR) T cell-based adoptive immunotherapy. Different methods used to facilitate efficient CRISPR delivery and gene editing in T cells are compared. The potential of genetic manipulation using CRISPR/Cas9 system to generate universal CAR T cells and potent T cells that are resistant to exhaustion and inhibition is explored. We also address the safety concerns associated with the use of CRISPR/Cas9 gene editing and provide potential solutions and future directions of CRISPR application in the field of CAR T cell immunotherapy. As an integration-free gene insertion method, CRISPR/Cas9 holds great promise as an efficient gene knock-in platform. Given the tremendous progress that has been made in the past few years, we believe that the CRISPR/Cas9 technology holds immense promise for advancing immunotherapy.

Targeted Delivery of CRISPR/Cas9-Mediated Cancer Gene Therapy via Liposome-Templated Hydrogel Nanoparticles.

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Chen, Zeming; Liu, Fuyao; Chen, Yanke; Liu, Jun; Wang, Xiaoying; Chen, Ann T; Deng, Gang; Zhang, Hongyi; Liu, Jie; Hong, Zhangyong; Zhou, Jiangbing

2017-12-08

Due to its simplicity, versatility, and high efficiency, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology has emerged as one of the most promising approaches for treatment of a variety of genetic diseases, including human cancers. However, further translation of CRISPR/Cas9 for cancer gene therapy requires development of safe approaches for efficient, highly specific delivery of both Cas9 and single guide RNA to tumors. Here, novel core-shell nanostructure, liposome-templated hydrogel nanoparticles (LHNPs) that are optimized for efficient codelivery of Cas9 protein and nucleic acids is reported. It is demonstrated that, when coupled with the minicircle DNA technology, LHNPs deliver CRISPR/Cas9 with efficiency greater than commercial agent Lipofectamine 2000 in cell culture and can be engineered for targeted inhibition of genes in tumors, including tumors the brain. When CRISPR/Cas9 targeting a model therapeutic gene, polo-like kinase 1 (PLK1), is delivered, LHNPs effectively inhibit tumor growth and improve tumor-bearing mouse survival. The results suggest LHNPs as versatile CRISPR/Cas9-delivery tool that can be adapted for experimentally studying the biology of cancer as well as for clinically translating cancer gene therapy.

CRISPR/Cas9 in Genome Editing and Beyond.

Science.gov (United States)

Wang, Haifeng; La Russa, Marie; Qi, Lei S

2016-06-02

The Cas9 protein (CRISPR-associated protein 9), derived from type II CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune systems, is emerging as a powerful tool for engineering the genome in diverse organisms. As an RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, and its development as a tool has made sequence-specific gene editing several magnitudes easier. The nuclease-deactivated form of Cas9 further provides a versatile RNA-guided DNA-targeting platform for regulating and imaging the genome, as well as for rewriting the epigenetic status, all in a sequence-specific manner. With all of these advances, we have just begun to explore the possible applications of Cas9 in biomedical research and therapeutics. In this review, we describe the current models of Cas9 function and the structural and biochemical studies that support it. We focus on the applications of Cas9 for genome editing, regulation, and imaging, discuss other possible applications and some technical considerations, and highlight the many advantages that CRISPR/Cas9 technology offers.

Functional Insights Revealed by the Kinetic Mechanism of CRISPR/Cas9.

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Raper, Austin T; Stephenson, Anthony A; Suo, Zucai

2018-02-28

The discovery of prokaryotic adaptive immunity prompted widespread use of the RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) endonuclease Cas9 for genetic engineering. However, its kinetic mechanism remains undefined, and details of DNA cleavage are poorly characterized. Here, we establish a kinetic mechanism of Streptococcus pyogenes Cas9 from guide-RNA binding through DNA cleavage and product release. Association of DNA to the binary complex of Cas9 and guide-RNA is rate-limiting during the first catalytic turnover, while DNA cleavage from a pre-formed ternary complex of Cas9, guide-RNA, and DNA is rapid. Moreover, an extremely slow release of DNA products essentially restricts Cas9 to be a single-turnover enzyme. By simultaneously measuring the contributions of the HNH and RuvC nuclease activities of Cas9 to DNA cleavage, we also uncovered the kinetic basis by which HNH conformationally regulates the RuvC cleavage activity. Together, our results provide crucial kinetic and functional details regarding Cas9 which will inform gene-editing experiments, guide future research to understand off-target DNA cleavage by Cas9, and aid in the continued development of Cas9 as a biotechnological tool.

CRISPR-Cas9 Structures and Mechanisms.

Science.gov (United States)

Jiang, Fuguo; Doudna, Jennifer A

2017-05-22

Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes. The use of CRISPR-Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)-CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.

The use of CRISPR/Cas associated technologies for cell transplant applications.

Science.gov (United States)

Cowan, Peter J

2016-10-01

In this review, I will summarize recent developments in the use of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) genome editing system for cell transplant applications, ranging from transplantation of corrected autologous patient stem cells to treat inherited diseases, to the tailoring of donor pigs for cell xenotransplantation. Rational engineering of the Cas9 nuclease to improve its specificity will also be discussed. Over the past year, CRISPR/Cas9 has been used in preclinical studies to correct mutations in a rapidly increasing spectrum of diseases including hematological, neuromuscular, and respiratory disorders. The growing popularity of CRISPR/Cas9 over earlier genome editing platforms is partly due to its ease of use and flexibility, which is evident from the success of complex manipulations such as specific deletion of up to 725 kb in patient-derived stem cells, and simultaneous disruption of up to 62 endogenous retrovirus loci in pig cells. In addition, high-fidelity variants of Cas9 with greatly increased specificity are now available. CRISPR/Cas9 is a fast-evolving technology that is likely to have a significant impact on autologous, allogeneic, and xenogeneic cell transplantation.

No evidence of inhibition of horizontal gene transfer by CRISPR-Cas on evolutionary timescales.

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Gophna, Uri; Kristensen, David M; Wolf, Yuri I; Popa, Ovidiu; Drevet, Christine; Koonin, Eugene V

2015-09-01

The CRISPR (clustered, regularly, interspaced, short, palindromic repeats)-Cas (CRISPR-associated genes) systems of archaea and bacteria provide adaptive immunity against viruses and other selfish elements and are believed to curtail horizontal gene transfer (HGT). Limiting acquisition of new genetic material could be one of the sources of the fitness cost of CRISPR-Cas maintenance and one of the causes of the patchy distribution of CRISPR-Cas among bacteria, and across environments. We sought to test the hypothesis that the activity of CRISPR-Cas in microbes is negatively correlated with the extent of recent HGT. Using three independent measures of HGT, we found no significant dependence between the length of CRISPR arrays, which reflects the activity of the immune system, and the estimated number of recent HGT events. In contrast, we observed a significant negative dependence between the estimated extent of HGT and growth temperature of microbes, which could be explained by the lower genetic diversity in hotter environments. We hypothesize that the relevant events in the evolution of resistance to mobile elements and proclivity for HGT, to which CRISPR-Cas systems seem to substantially contribute, occur on the population scale rather than on the timescale of species evolution.

CRISPR Primer Designer: Design primers for knockout and chromosome imaging CRISPR-Cas system.

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Yan, Meng; Zhou, Shi-Rong; Xue, Hong-Wei

2015-07-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated system enables biologists to edit genomes precisely and provides a powerful tool for perturbing endogenous gene regulation, modulation of epigenetic markers, and genome architecture. However, there are concerns about the specificity of the system, especially the usages of knocking out a gene. Previous designing tools either were mostly built-in websites or ran as command-line programs, and none of them ran locally and acquired a user-friendly interface. In addition, with the development of CRISPR-derived systems, such as chromosome imaging, there were still no tools helping users to generate specific end-user spacers. We herein present CRISPR Primer Designer for researchers to design primers for CRISPR applications. The program has a user-friendly interface, can analyze the BLAST results by using multiple parameters, score for each candidate spacer, and generate the primers when using a certain plasmid. In addition, CRISPR Primer Designer runs locally and can be used to search spacer clusters, and exports primers for the CRISPR-Cas system-based chromosome imaging system. © 2014 Institute of Botany, Chinese Academy of Sciences.

Imipenem represses CRISPR-Cas interference of DNA acquisition through H-NS stimulation in Klebsiella pneumoniae.

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Lin, Tzu-Lung; Pan, Yi-Jiun; Hsieh, Pei-Fang; Hsu, Chun-Ru; Wu, Meng-Chuan; Wang, Jin-Town

2016-08-17

Analysis of the genome of Klebsiella pneumoniae NTUH-K2044 strain revealed the presence of two clustered regularly interspaced short palindromic repeats (CRISPR) arrays separated with CRISPR-associated (cas) genes. Carbapenem-resistant K. pneumoniae isolates were observed to be less likely to have CRISPR-Cas than sensitive strains (5/85 vs. 22/132). Removal of the transcriptional repressor, H-NS, was shown to prevent the transformation of plasmids carrying a spacer and putative proto-spacer adjacent motif (PAM). The CRISPR-Cas system also decreased pUC-4K plasmid stability, resulting in plasmid loss from the bacteria with acquisition of new spacers. Analysis of the acquired proto-spacers in pUC-4K indicated that 5'-TTN-3' was the preferred PAM in K. pneumoniae. Treatment of cells by imipenem induced hns expression, thereby decreasing cas3 expression and consequently repressed CRISPR-Cas activity resulted in increase of plasmid stability. In conclusion, NTUH-K2044 CRISPR-Cas contributes to decrease of plasmid transformation and stability. Through repression of CRISPR-Cas activity by induced H-NS, bacteria might be more able to acquire DNA to confront the challenge of imipenem.

The role of CRISPR-Cas systems in virulence of pathogenic bacteria.

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Louwen, Rogier; Staals, Raymond H J; Endtz, Hubert P; van Baarlen, Peter; van der Oost, John

2014-03-01

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are present in many bacterial and archaeal genomes. Since the discovery of the typical CRISPR loci in the 1980s, well before their physiological role was revealed, their variable sequences have been used as a complementary typing tool in diagnostic, epidemiologic, and evolutionary analyses of prokaryotic strains. The discovery that CRISPR spacers are often identical to sequence fragments of mobile genetic elements was a major breakthrough that eventually led to the elucidation of CRISPR-Cas as an adaptive immunity system. Key elements of this unique prokaryotic defense system are small CRISPR RNAs that guide nucleases to complementary target nucleic acids of invading viruses and plasmids, generally followed by the degradation of the invader. In addition, several recent studies have pointed at direct links of CRISPR-Cas to regulation of a range of stress-related phenomena. An interesting example concerns a pathogenic bacterium that possesses a CRISPR-associated ribonucleoprotein complex that may play a dual role in defense and/or virulence. In this review, we describe recently reported cases of potential involvement of CRISPR-Cas systems in bacterial stress responses in general and bacterial virulence in particular.

A CRISPR Path to Engineering New Genetic Mouse Models for Cardiovascular Research.

Science.gov (United States)

Miano, Joseph M; Zhu, Qiuyu Martin; Lowenstein, Charles J

2016-06-01

Previous efforts to target the mouse genome for the addition, subtraction, or substitution of biologically informative sequences required complex vector design and a series of arduous steps only a handful of laboratories could master. The facile and inexpensive clustered regularly interspaced short palindromic repeats (CRISPR) method has now superseded traditional means of genome modification such that virtually any laboratory can quickly assemble reagents for developing new mouse models for cardiovascular research. Here, we briefly review the history of CRISPR in prokaryotes, highlighting major discoveries leading to its formulation for genome modification in the animal kingdom. Core components of CRISPR technology are reviewed and updated. Practical pointers for 2-component and 3-component CRISPR editing are summarized with many applications in mice including frameshift mutations, deletion of enhancers and noncoding genes, nucleotide substitution of protein-coding and gene regulatory sequences, incorporation of loxP sites for conditional gene inactivation, and epitope tag integration. Genotyping strategies are presented and topics of genetic mosaicism and inadvertent targeting discussed. Finally, clinical applications and ethical considerations are addressed as the biomedical community eagerly embraces this astonishing innovation in genome editing to tackle previously intractable questions. © 2016 American Heart Association, Inc.

Restriction-modification mediated barriers to exogenous DNA uptake and incorporation employed by Prevotella intermedia.

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Johnston, Christopher D; Skeete, Chelsey A; Fomenkov, Alexey; Roberts, Richard J; Rittling, Susan R

2017-01-01

Prevotella intermedia, a major periodontal pathogen, is increasingly implicated in human respiratory tract and cystic fibrosis lung infections. Nevertheless, the specific mechanisms employed by this pathogen remain only partially characterized and poorly understood, largely due to its total lack of genetic accessibility. Here, using Single Molecule, Real-Time (SMRT) genome and methylome sequencing, bisulfite sequencing, in addition to cloning and restriction analysis, we define the specific genetic barriers to exogenous DNA present in two of the most widespread laboratory strains, P. intermedia ATCC 25611 and P. intermedia Strain 17. We identified and characterized multiple restriction-modification (R-M) systems, some of which are considerably divergent between the two strains. We propose that these R-M systems are the root cause of the P. intermedia transformation barrier. Additionally, we note the presence of conserved Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems in both strains, which could provide a further barrier to exogenous DNA uptake and incorporation. This work will provide a valuable resource during the development of a genetic system for P. intermedia, which will be required for fundamental investigation of this organism's physiology, metabolism, and pathogenesis in human disease.

Editing Transgenic DNA Components by Inducible Gene Replacement in Drosophila melanogaster

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Lin, Chun-Chieh; Potter, Christopher J.

2016-01-01

Gene conversions occur when genomic double-strand DNA breaks (DSBs) trigger unidirectional transfer of genetic material from a homologous template sequence. Exogenous or mutated sequence can be introduced through this homology-directed repair (HDR). We leveraged gene conversion to develop a method for genomic editing of existing transgenic insertions in Drosophila melanogaster. The clustered regularly-interspaced palindromic repeats (CRISPR)/Cas9 system is used in the homology assisted CRISPR knock-in (HACK) method to induce DSBs in a GAL4 transgene, which is repaired by a single-genomic transgenic construct containing GAL4 homologous sequences flanking a T2A-QF2 cassette. With two crosses, this technique converts existing GAL4 lines, including enhancer traps, into functional QF2 expressing lines. We used HACK to convert the most commonly-used GAL4 lines (labeling tissues such as neurons, fat, glia, muscle, and hemocytes) to QF2 lines. We also identified regions of the genome that exhibited differential efficiencies of HDR. The HACK technique is robust and readily adaptable for targeting and replacement of other genomic sequences, and could be a useful approach to repurpose existing transgenes as new genetic reagents become available. PMID:27334272

Single cell genomics indicates horizontal gene transfer and viral infections in a deep subsurface Firmicutes population

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Jessica eLabonté

2015-04-01

Full Text Available A major fraction of Earth's prokaryotic biomass dwells in the deep subsurface, where cellular abundances per volume of sample are lower, metabolism is slower, and generation times are longer than those in surface terrestrial and marine environments. How these conditions impact biotic interactions and evolutionary processes is largely unknown. Here we employed single cell genomics to analyze cell-to-cell genome content variability and signatures of horizontal gene transfer (HGT and viral infections in five cells of Candidatus Desulforudis audaxviator, which were collected from a three km-deep fracture water in the 2.9 Ga-old Witwatersrand Basin of South Africa. Between 0 and 32 % of genes recovered from single cells were not present in the original, metagenomic assembly of Desulforudis, which was obtained from a neighboring subsurface fracture. We found a transposable prophage, a retron, multiple clustered regularly interspaced short palindromic repeats (CRISPRs and restriction-modification systems, and an unusually high frequency of transposases in the analyzed single cell genomes. This indicates that recombination, HGT and viral infections are prevalent evolutionary events in the studied population of microorganisms inhabiting a highly stable deep subsurface environment.

Advancing chimeric antigen receptor T cell therapy with CRISPR/Cas9

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Jiangtao Ren

2017-04-01

Full Text Available ABSTRACT The clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR-associated 9 (CRISPR/Cas9 system, an RNA-guided DNA targeting technology, is triggering a revolution in the field of biology. CRISPR/Cas9 has demonstrated great potential for genetic manipulation. In this review, we discuss the current development of CRISPR/Cas9 technologies for therapeutic applications, especially chimeric antigen receptor (CAR T cell-based adoptive immunotherapy. Different methods used to facilitate efficient CRISPR delivery and gene editing in T cells are compared. The potential of genetic manipulation using CRISPR/Cas9 system to generate universal CAR T cells and potent T cells that are resistant to exhaustion and inhibition is explored. We also address the safety concerns associated with the use of CRISPR/Cas9 gene editing and provide potential solutions and future directions of CRISPR application in the field of CAR T cell immunotherapy. As an integration-free gene insertion method, CRISPR/Cas9 holds great promise as an efficient gene knock-in platform. Given the tremendous progress that has been made in the past few years, we believe that the CRISPR/Cas9 technology holds immense promise for advancing immunotherapy.

Genome editing technologies to fight infectious diseases.

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Trevisan, Marta; Palù, Giorgio; Barzon, Luisa

2017-11-01

Genome editing by programmable nucleases represents a promising tool that could be exploited to develop new therapeutic strategies to fight infectious diseases. These nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) and homing endonucleases, are molecular scissors that can be targeted at predetermined loci in order to modify the genome sequence of an organism. Areas covered: By perturbing genomic DNA at predetermined loci, programmable nucleases can be used as antiviral and antimicrobial treatment. This approach includes targeting of essential viral genes or viral sequences able, once mutated, to inhibit viral replication; repurposing of CRISPR-Cas9 system for lethal self-targeting of bacteria; targeting antibiotic-resistance and virulence genes in bacteria, fungi, and parasites; engineering arthropod vectors to prevent vector-borne infections. Expert commentary: While progress has been done in demonstrating the feasibility of using genome editing as antimicrobial strategy, there are still many hurdles to overcome, such as the risk of off-target mutations, the raising of escape mutants, and the inefficiency of delivery methods, before translating results from preclinical studies into clinical applications.

Genome Editing with Crispr-Cas9 Systems: Basic Research and Clinical Applications

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Anna Meiliana

2017-04-01

Full Text Available BACKGROUND: Recently established genome editing technologies will open new avenues for biological research and development. Human genome editing is a powerful tool which offers great scientific and therapeutic potential. CONTENT: Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR/CRISPRassociated protein 9 (Cas9 technology is revolutionizing the gene function studies and possibly will give rise to an entirely new degree of therapeutics for a large range of diseases. Prompt advances in the CRISPR/Cas9 technology, as well as delivery modalities for gene therapy applications, are dismissing the barriers to the clinical translation of this technology. Many studies conducted showed promising results, but as current available technologies for evaluating off-target gene modification, several elements must be addressed to validate the safety of the CRISPR/Cas9 platform for clinical application, as the ethical implication as well. SUMMARY: The CRISPR/Cas9 system is a powerful genome editing technology with the potential to create a variety of novel therapeutics for a range of diseases, many of which are currently untreatable. KEYWORDS: genome editing, CRISPR-Cas, guideRNA, DSB, ZFNs, TALEN

In Vitro and In Vivo Synergistic Therapeutic Effect of Cisplatin with Human Papillomavirus16 E6/E7 CRISPR/Cas9 on Cervical Cancer Cell Line

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Shuai Zhen

2016-12-01

Full Text Available PURPOSE: Human papillomavirus (HPV type 16 is one of the major etiologic factors of cervical cancer. Our study aims to investigate the potentiality of the antiviral clustered regularly interspaced short palindromic repeat (CRISPR/CRISPR-associated Cas9 system (CRISPR/Cas9 targeting the E6 and E7 oncogenes of HPV16 as a potential chemosensitizer of cisplatin (cis-diaminedichloroplatinum II; CDDP for cervical cancer. METHODS: Specifically, the therapeutic efficacy of combination of CDDP and HPV16 E6 + E7-CRISPR/Cas9 was assessed in cervical cancer cells and cervical cancer xenograft models. RESULTS: In vitro experiments showed that long-term exposure of SiHa cells to the HPV16 E6 + E7-CRISPR/Cas9 induced apoptosis, and its pro-apoptosis effect became more obvious when combined with CDDP. In vivo study found the efficacy of the combination of HPV16 E6 + E7-CRISPR/Cas9 and CDDP were superior to either of the treatments in term of apoptosis induction and metastasis inhibition. CONCLUSION: Collectively, our results suggested that HPV16 E6 + E7-CRISPR/Cas9 could be an effective sensitizer of CDDP chemotherapy in cervical cancer.

Gene Disruption Technologies Have the Potential to Transform Stored Product Insect Pest Control.

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Perkin, Lindsey C; Adrianos, Sherry L; Oppert, Brenda

2016-09-19

Stored product insects feed on grains and processed commodities manufactured from grain post-harvest, reducing the nutritional value and contaminating food. Currently, the main defense against stored product insect pests is the pesticide fumigant phosphine. Phosphine is highly toxic to all animals, but is the most effective and economical control method, and thus is used extensively worldwide. However, many insect populations have become resistant to phosphine, in some cases to very high levels. New, environmentally benign and more effective control strategies are needed for stored product pests. RNA interference (RNAi) may overcome pesticide resistance by targeting the expression of genes that contribute to resistance in insects. Most data on RNAi in stored product insects is from the coleopteran genetic model, Tribolium castaneum, since it has a strong RNAi response via injection of double stranded RNA (dsRNA) in any life stage. Additionally, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has been suggested as a potential resource for new pest control strategies. In this review we discuss background information on both gene disruption technologies and summarize the advances made in terms of molecular pest management in stored product insects, mainly T. castaneum, as well as complications and future needs.

Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system.

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Sinkunas, Tomas; Gasiunas, Giedrius; Fremaux, Christophe; Barrangou, Rodolphe; Horvath, Philippe; Siksnys, Virginijus

2011-04-06

Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA-guided interference with virus proliferation. Cas3 contains N-terminal HD phosphohydrolase and C-terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single-stranded DNA (ssDNA)-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP-independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation-of-function mutants (ATPase(+)/nuclease(-) and ATPase(-)/nuclease(+)) were obtained by site-directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade-crRNA complex targeting foreign DNA.

The Conspicuity of CRISPR-Cpf1 System as a Significant Breakthrough in Genome Editing.

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Bayat, Hadi; Modarressi, Mohammad Hossein; Rahimpour, Azam

2018-01-01

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) is a microbial adaptive immune system. CRISPR-Cas systems are classified into two main classes and six types. Cpf1 is a putative type V (class II) CRISPR effector, which has revolutionized the genome editing approaches through multiple distinct features such as using T-rich protospacer-adjacent motif, applying a short guide RNA lacking trans-activating crRNA, introducing a staggered double-strand break, and possessing RNA processing activity in addition to DNA nuclease activity. In the present review, we attempt to highlight most recent advances in CRISPR-Cpf1 (CRISPR-Cas12a) system in particular, considering ground expeditions of the nature and the biology of this system, introducing novel Cpf1 variants that have broadened the versatility and feasibility of CRISPR-Cpf1 system, and lastly the great impact of the CRISPR-Cpf1 system on the manipulation of the genome of prokaryotic, mammalian, and plant models is summarized. With regard to recent developments in utilizing the CRISPR-Cpf1 system in genome editing of various organisms, it can be concluded with confidence that this system is a reliable molecular toolbox of genome editing approaches.

A CRISPR Path to Engineering New Genetic Mouse Models for Cardiovascular Research

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Miano, Joseph M.; Zhu, Qiuyu Martin; Lowenstein, Charles J.

2016-01-01

Previous efforts to target the mouse genome for the addition, subtraction, or substitution of biologically informative sequences required complex vector design and a series of arduous steps only a handful of labs could master. The facile and inexpensive clustered regularly interspaced short palindromic repeats (CRISPR) method has now superseded traditional means of genome modification such that virtually any lab can quickly assemble reagents for developing new mouse models for cardiovascular research. Here we briefly review the history of CRISPR in prokaryotes, highlighting major discoveries leading to its formulation for genome modification in the animal kingdom. Core components of CRISPR technology are reviewed and updated. Practical pointers for two-component and three-component CRISPR editing are summarized with a number of applications in mice including frameshift mutations, deletion of enhancers and non-coding genes, nucleotide substitution of protein-coding and gene regulatory sequences, incorporation of loxP sites for conditional gene inactivation, and epitope tag integration. Genotyping strategies are presented and topics of genetic mosaicism and inadvertent targeting discussed. Finally, clinical applications and ethical considerations are addressed as the biomedical community eagerly embraces this astonishing innovation in genome editing to tackle previously intractable questions. PMID:27102963

Haloarcula hispanica CRISPR authenticates PAM of a target sequence to prime discriminative adaptation.

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Li, Ming; Wang, Rui; Xiang, Hua

2014-06-01

The prokaryotic immune system CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated genes) adapts to foreign invaders by acquiring their short deoxyribonucleic acid (DNA) fragments as spacers, which guide subsequent interference to foreign nucleic acids based on sequence matching. The adaptation mechanism avoiding acquiring 'self' DNA fragments is poorly understood. In Haloarcula hispanica, we previously showed that CRISPR adaptation requires being primed by a pre-existing spacer partially matching the invader DNA. Here, we further demonstrate that flanking a fully-matched target sequence, a functional PAM (protospacer adjacent motif) is still required to prime adaptation. Interestingly, interference utilizes only four PAM sequences, whereas adaptation-priming tolerates as many as 23 PAM sequences. This relaxed PAM selectivity explains how adaptation-priming maximizes its tolerance of PAM mutations (that escape interference) while avoiding mis-targeting the spacer DNA within CRISPR locus. We propose that the primed adaptation, which hitches and cooperates with the interference pathway, distinguishes target from non-target by CRISPR ribonucleic acid guidance and PAM recognition. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Numerical taxonomy of the genus Pestivirus based on palindromic nucleotide substitutions in the 5' untranslated region.

Science.gov (United States)

Giangaspero, Massimo; Harasawa, Ryô

2007-12-01

The palindromic nucleotide substitutions (PNS) at the three variable loci (V1, V2 and V3) in the 5' untranslated region (UTR) of Pestivirus RNA have been considered for taxonomical segregation of species, through the evaluation of 430 genomic sequences. On the basis of qualitative and quantitative secondary structure characteristics, six species have been identified: Bovine viral diarrhea virus 1 (BVDV-1), Bovine viral diarrhea virus 2 (BVDV-2), Classical swine fever virus (CSFV), Border disease virus (BDV), the tentative species Giraffe and a new proposed taxon named Pronghorn. The first step was qualitative and consisted in the characterization of the different positions of the three stems and loops in the 5' UTR sequences of all the strains under consideration belonging to the genus. Secondary structure sequences showing divergent base-pair combinations have been aligned for comparison. Palindromic positions have been characterized according to changes in nucleotide base-pairs identifying low-variable positions (LVP) including base-pairs present in less than 80% of the genus. The second step was quantitative, allowing the identification of genomic groups by clustering the base-pair combinations according to LVP. Relatedness among types was evaluated to identify homogeneous groups. Cross comparisons between types within the genus have been evaluated by computing the divergence percentage thus clarifying borderline and multirelated sequences.

Trapping of palindromic ligands within native transthyretin prevents amyloid formation

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Kolstoe, Simon E.; Mangione, Palma P.; Bellotti, Vittorio; Taylor, Graham W.; Tennent, Glenys A.; Deroo, Stéphanie; Morrison, Angus J.; Cobb, Alexander J. A.; Coyne, Anthony; McCammon, Margaret G.; Warner, Timothy D.; Mitchell, Jane; Gill, Raj; Smith, Martin D.; Ley, Steven V.; Robinson, Carol V.; Wood, Stephen P.; Pepys, Mark B.

2010-01-01

Transthyretin (TTR) amyloidosis is a fatal disease for which new therapeutic approaches are urgently needed. We have designed two palindromic ligands, 2,2'-(4,4'-(heptane-1,7-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (mds84) and 2,2'-(4,4'-(undecane-1,11-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (4ajm15), that are rapidly bound by native wild-type TTR in whole serum and even more avidly by amyloidogenic TTR variants. One to one stoichiometry, demonstrable in solution and by MS, was confirmed by X-ray crystallographic analysis showing simultaneous occupation of both T4 binding sites in each tetrameric TTR molecule by the pair of ligand head groups. Ligand binding by native TTR was irreversible under physiological conditions, and it stabilized the tetrameric assembly and inhibited amyloidogenic aggregation more potently than other known ligands. These superstabilizers are orally bioavailable and exhibit low inhibitory activity against cyclooxygenase (COX). They offer a promising platform for development of drugs to treat and prevent TTR amyloidosis. PMID:21059958

Essential Structural and Functional Roles of the Cmr4 Subunit in RNA Cleavage by the Cmr CRISPR-Cas Complex

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Nancy F. Ramia

2014-12-01

Full Text Available Summary: The Cmr complex is the multisubunit effector complex of the type III-B clustered regularly interspaced short palindromic repeats (CRISPR-Cas immune system. The Cmr complex recognizes a target RNA through base pairing with the integral CRISPR RNA (crRNA and cleaves the target at multiple regularly spaced locations within the complementary region. To understand the molecular basis of the function of this complex, we have assembled information from electron microscopic and X-ray crystallographic structural studies and mutagenesis of a complete Pyrococcus furiosus Cmr complex. Our findings reveal that four helically packed Cmr4 subunits, which make up the backbone of the Cmr complex, act as a platform to support crRNA binding and target RNA cleavage. Interestingly, we found a hook-like structural feature associated with Cmr4 that is likely the site of target RNA binding and cleavage. Our results also elucidate analogies in the mechanisms of crRNA and target molecule binding by the distinct Cmr type III-A and Cascade type I-E complexes. : Ramia et al. show that the helical core of the type III-B Cmr CRISPR-Cas effector complex, made up of multiple Cmr4 subunits, forms the platform for a corresponding number of cleavages of the target RNA. Comparison with the type I-E Cascade structure reveals strikingly similar mechanisms of crRNA and target binding.

Specific interaction of the nonstructural protein NS1 of minute virus of mice (MVM) with [ACCA](2) motifs in the centre of the right-end MVM DNA palindrome induces hairpin-primed viral DNA replication.

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Willwand, Kurt; Moroianu, Adela; Hörlein, Rita; Stremmel, Wolfgang; Rommelaere, Jean

2002-07-01

The linear single-stranded DNA genome of minute virus of mice (MVM) is replicated via a double-stranded replicative form (RF) intermediate DNA. Amplification of viral RF DNA requires the structural transition of the right-end palindrome from a linear duplex into a double-hairpin structure, which serves for the repriming of unidirectional DNA synthesis. This conformational transition was found previously to be induced by the MVM nonstructural protein NS1. Elimination of the cognate NS1-binding sites, [ACCA](2), from the central region of the right-end palindrome next to the axis of symmetry was shown to markedly reduce the efficiency of hairpin-primed DNA replication, as measured in a reconstituted in vitro replication system. Thus, [ACCA](2) sequence motifs are essential as NS1-binding elements in the context of the structural transition of the right-end MVM palindrome.

Conformational Diversity of Single-Stranded DNA from Bacterial Repetitive Extragenic Palindromes: Implications for the DNA Recognition Elements of Transposases

Czech Academy of Sciences Publication Activity Database

Charnavets, Tatsiana; Nunvář, Jaroslav; Nečasová, Iva; Voelker, J.; Breslauer, K.J.; Schneider, Bohdan

2015-01-01

Roč. 103, č. 10 (2015), s. 585-596 ISSN 0006-3525 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109; GA ČR GAP305/12/1801; GA MŠk(CZ) EE2.3.30.0020 Institutional support: RVO:86652036 Keywords : bacterial repetitive extragenic palindromes (REP) * circular dichroism spectroscopy * REP associated tyrosine transposases (RAYTs) Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.248, year: 2015

A PNPase Dependent CRISPR System in Listeria

Science.gov (United States)

Sesto, Nina; Touchon, Marie; Andrade, José Marques; Kondo, Jiro; Rocha, Eduardo P. C.; Arraiano, Cecilia Maria; Archambaud, Cristel; Westhof, Éric; Romby, Pascale; Cossart, Pascale

2014-01-01

The human bacterial pathogen Listeria monocytogenes is emerging as a model organism to study RNA-mediated regulation in pathogenic bacteria. A class of non-coding RNAs called CRISPRs (clustered regularly interspaced short palindromic repeats) has been described to confer bacterial resistance against invading bacteriophages and conjugative plasmids. CRISPR function relies on the activity of CRISPR associated (cas) genes that encode a large family of proteins with nuclease or helicase activities and DNA and RNA binding domains. Here, we characterized a CRISPR element (RliB) that is expressed and processed in the L. monocytogenes strain EGD-e, which is completely devoid of cas genes. Structural probing revealed that RliB has an unexpected secondary structure comprising basepair interactions between the repeats and the adjacent spacers in place of canonical hairpins formed by the palindromic repeats. Moreover, in contrast to other CRISPR-Cas systems identified in Listeria, RliB-CRISPR is ubiquitously present among Listeria genomes at the same genomic locus and is never associated with the cas genes. We showed that RliB-CRISPR is a substrate for the endogenously encoded polynucleotide phosphorylase (PNPase) enzyme. The spacers of the different Listeria RliB-CRISPRs share many sequences with temperate and virulent phages. Furthermore, we show that a cas-less RliB-CRISPR lowers the acquisition frequency of a plasmid carrying the matching protospacer, provided that trans encoded cas genes of a second CRISPR-Cas system are present in the genome. Importantly, we show that PNPase is required for RliB-CRISPR mediated DNA interference. Altogether, our data reveal a yet undescribed CRISPR system whose both processing and activity depend on PNPase, highlighting a new and unexpected function for PNPase in “CRISPRology”. PMID:24415952

Diverse Regular Employees and Non-regular Employment (Japanese)

OpenAIRE

MORISHIMA Motohiro

2011-01-01

Currently there are high expectations for the introduction of policies related to diverse regular employees. These policies are a response to the problem of disparities between regular and non-regular employees (part-time, temporary, contract and other non-regular employees) and will make it more likely that workers can balance work and their private lives while companies benefit from the advantages of regular employment. In this paper, I look at two issues that underlie this discussion. The ...

Investigation of potential targets of Porphyromonas CRISPRs among the genomes of Porphyromonas species.

Science.gov (United States)

Watanabe, Takayasu; Shibasaki, Masaki; Maruyama, Fumito; Sekizaki, Tsutomu; Nakagawa, Ichiro

2017-01-01

The oral bacterial species Porphyromonas gingivalis, a periodontal pathogen, has plastic genomes that may be driven by homologous recombination with exogenous deoxyribonucleic acid (DNA) that is incorporated by natural transformation and conjugation. However, bacteriophages and plasmids, both of which are main resources of exogenous DNA, do not exist in the known P. gingivalis genomes. This could be associated with an adaptive immunity system conferred by clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (cas) genes in P. gingivalis as well as innate immune systems such as a restriction-modification system. In a previous study, few immune targets were predicted for P. gingivalis CRISPR/Cas. In this paper, we analyzed 51 P. gingivalis genomes, which were newly sequenced, and publicly available genomes of 13 P. gingivalis and 46 other Porphyromonas species. We detected 6 CRISPR/Cas types (classified by sequence similarity of repeat) in P. gingivalis and 12 other types in the remaining species. The Porphyromonas CRISPR spacers with potential targets in the genus Porphyromonas were approximately 23 times more abundant than those with potential targets in other genus taxa (1,720/6,896 spacers vs. 74/6,896 spacers). Porphyromonas CRISPR/Cas may be involved in genome plasticity by exhibiting selective interference against intra- and interspecies nucleic acids.

RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors

KAUST Repository

Piatek, Agnieszka Anna

2014-11-14

Targeted genomic regulation is a powerful approach to accelerate trait discovery and development in agricultural biotechnology. Bacteria and archaea use clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) regulatory systems for adaptive molecular immunity against foreign nucleic acids introduced by invading phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing in many cell types and organisms. A recent study used the catalytically inactive Cas9 (dCas9) protein combined with guide-RNAs (gRNAs) as a DNA-targeting platform to modulate gene expression in bacterial, yeast, and human cells. Here, we modified this DNA-targeting platform for targeted transcriptional regulation in planta by developing chimeric dCas9-based transcriptional activators and repressors. To generate transcriptional activators, we fused the dCas9 C-terminus with the activation domains of EDLL and TAL effectors. To generate a transcriptional repressor, we fused the dCas9 C-terminus with the SRDX repression domain. Our data demonstrate that dCas9 fusion with the EDLL activation domain (dCas9:EDLL) and the TAL activation domain (dCas9:TAD), guided by gRNAs complementary to selected promoter elements, induce strong transcriptional activation on Bs3

Targeted genome regulation via synthetic programmable transcriptional regulators

KAUST Repository

Piatek, Agnieszka Anna

2016-04-19

Regulation of gene transcription controls cellular functions and coordinates responses to developmental, physiological and environmental cues. Precise and efficient molecular tools are needed to characterize the functions of single and multiple genes in linear and interacting pathways in a native context. Modular DNA-binding domains from zinc fingers (ZFs) and transcriptional activator-like proteins (TALE) are amenable to bioengineering to bind DNA target sequences of interest. As a result, ZF and TALE proteins were used to develop synthetic programmable transcription factors. However, these systems are limited by the requirement to re-engineer proteins for each new target sequence. The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated 9 (Cas9) genome editing tool was recently repurposed for targeted transcriptional regulation by inactivation of the nuclease activity of Cas9. Due to the facile engineering, simplicity, precision and amenability to library construction, the CRISPR/Cas9 system is poised to revolutionize the functional genomics field across diverse eukaryotic species. In this review, we discuss the development of synthetic customizable transcriptional regulators and provide insights into their current and potential applications, with special emphasis on plant systems, in characterization of gene functions, elucidation of molecular mechanisms and their biotechnological applications. © 2016 Informa UK Limited, trading as Taylor & Francis Group

Progress and Prospects of CRISPR/Cas Systems in Insects and Other Arthropods

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Dan Sun

2017-09-01

Full Text Available Clustered regularly interspaced short palindromic repeats (CRISPR and the CRISPR-associated gene Cas9 represent an invaluable system for the precise editing of genes in diverse species. The CRISPR/Cas9 system is an adaptive mechanism that enables bacteria and archaeal species to resist invading viruses and phages or plasmids. Compared with zinc finger nucleases and transcription activator-like effector nucleases, the CRISPR/Cas9 system has the advantage of requiring less time and effort. This efficient technology has been used in many species, including diverse arthropods that are relevant to agriculture, forestry, fisheries, and public health; however, there is no review that systematically summarizes its successful application in the editing of both insect and non-insect arthropod genomes. Thus, this paper seeks to provide a comprehensive and impartial overview of the progress of the CRISPR/Cas9 system in different arthropods, reviewing not only fundamental studies related to gene function exploration and experimental optimization but also applied studies in areas such as insect modification and pest control. In addition, we also describe the latest research advances regarding two novel CRISPR/Cas systems (CRISPR/Cpf1 and CRISPR/C2c2 and discuss their future prospects for becoming crucial technologies in arthropods.

Genome engineering in ophthalmology: Application of CRISPR/Cas to the treatment of eye disease.

Science.gov (United States)

Hung, Sandy S C; McCaughey, Tristan; Swann, Olivia; Pébay, Alice; Hewitt, Alex W

2016-07-01

The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR-associated protein (Cas) system has enabled an accurate and efficient means to edit the human genome. Rapid advances in this technology could results in imminent clinical application, and with favourable anatomical and immunological profiles, ophthalmic disease will be at the forefront of such work. There have been a number of breakthroughs improving the specificity and efficacy of CRISPR/Cas-mediated genome editing. Similarly, better methods to identify off-target cleavage sites have also been developed. With the impending clinical utility of CRISPR/Cas technology, complex ethical issues related to the regulation and management of the precise applications of human gene editing must be considered. This review discusses the current progress and recent breakthroughs in CRISPR/Cas-based gene engineering, and outlines some of the technical issues that must be addressed before gene correction, be it in vivo or in vitro, is integrated into ophthalmic care. We outline a clinical pipeline for CRISPR-based treatments of inherited eye diseases and provide an overview of the important ethical implications of gene editing and how these may influence the future of this technology. Copyright © 2016 Elsevier Ltd. All rights reserved.

CRISPR/Cas9-Mediated Correction of the FANCD1 Gene in Primary Patient Cells

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Karolina Skvarova Kramarzova

2017-06-01

Full Text Available Fanconi anemia (FA is an inherited condition characterized by impaired DNA repair, physical anomalies, bone marrow failure, and increased incidence of malignancy. Gene editing holds great potential to precisely correct the underlying genetic cause such that gene expression remains under the endogenous control mechanisms. This has been accomplished to date only in transformed cells or their reprogrammed induced pluripotent stem cell counterparts; however, it has not yet been reported in primary patient cells. Here we show the ability to correct a mutation in Fanconi anemia D1 (FANCD1 primary patient fibroblasts. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9 system was employed to target and correct a FANCD1 gene deletion. Homologous recombination using an oligonucleotide donor was achieved and a pure population of modified cells was obtained by using inhibitors of poly adenosine diphosphate-ribose polymerase (poly ADP-ribose polymerase. FANCD1 function was restored and we did not observe any promiscuous cutting of the CRISPR/Cas9 at off target sites. This consideration is crucial in the context of the pre-malignant FA phenotype. Altogether we show the ability to correct a patient mutation in primary FANCD1 cells in a precise manner. These proof of principle studies support expanded application of gene editing for FA.

The Impact of CRISPR/Cas9 Technology on Cardiac Research: From Disease Modelling to Therapeutic Approaches

Science.gov (United States)

Pramstaller, Peter P.; Hicks, Andrew A.; Rossini, Alessandra

2017-01-01

Genome-editing technology has emerged as a powerful method that enables the generation of genetically modified cells and organisms necessary to elucidate gene function and mechanisms of human diseases. The clustered regularly interspaced short palindromic repeats- (CRISPR-) associated 9 (Cas9) system has rapidly become one of the most popular approaches for genome editing in basic biomedical research over recent years because of its simplicity and adaptability. CRISPR/Cas9 genome editing has been used to correct DNA mutations ranging from a single base pair to large deletions in both in vitro and in vivo model systems. CRISPR/Cas9 has been used to increase the understanding of many aspects of cardiovascular disorders, including lipid metabolism, electrophysiology and genetic inheritance. The CRISPR/Cas9 technology has been proven to be effective in creating gene knockout (KO) or knockin in human cells and is particularly useful for editing induced pluripotent stem cells (iPSCs). Despite these progresses, some biological, technical, and ethical issues are limiting the therapeutic potential of genome editing in cardiovascular diseases. This review will focus on various applications of CRISPR/Cas9 genome editing in the cardiovascular field, for both disease research and the prospect of in vivo genome-editing therapies in the future. PMID:29434642

Phage typing or CRISPR typing for epidemiological surveillance of Salmonella Typhimurium?

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Mohammed, Manal

2017-11-07

Salmonella Typhimurium is the most dominant Salmonella serovar around the world. It is associated with foodborne gastroenteritis outbreaks but has recently been associated with invasive illness and deaths. Characterization of S. Typhimurium is therefore very crucial for epidemiological surveillance. Phage typing has been used for decades for subtyping of S. Typhimurium to determine the epidemiological relation among isolates. Recent studies however have suggested that high throughput clustered regular interspaced short palindromic repeats (CRISPR) typing has the potential to replace phage typing. This study aimed to determine the efficacy of high-throughput CRISPR typing over conventional phage typing in epidemiological surveillance and outbreak investigation of S. Typhimurium. In silico analysis of whole genome sequences (WGS) of well-documented phage types of S. Typhimurium reveals the presence of different CRISPR type among strains belong to the same phage type. Furthermore, different phage types of S. Typhimurium share identical CRISPR type. Interestingly, identical spacers were detected among outbreak and non-outbreak associated DT8 strains of S. Typhimurium. Therefore, CRISPR typing is not useful for the epidemiological surveillance and outbreak investigation of S. Typhimurium and phage typing, until it is replaced by WGS, is still the gold standard method for epidemiological surveillance of S. Typhimurium.

Applications of the CRISPR-Cas9 system in kidney research.

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Higashijima, Yoshiki; Hirano, Seiichi; Nangaku, Masaomi; Nureki, Osamu

2017-08-01

The recently discovered clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) is an RNA-guided DNA nuclease, and has been harnessed for the development of simple, efficient, and relatively inexpensive technologies to precisely manipulate the genomic information in virtually all cell types and organisms. The CRIPSR-Cas9 systems have already been effectively used to disrupt multiple genes simultaneously, create conditional alleles, and generate reporter proteins, even in vivo. The ability of Cas9 to target a specific genomic region has also been exploited for various applications, such as transcriptional regulation, epigenetic control, and chromosome labeling. Here we first describe the molecular mechanism of the RNA-guided DNA targeting by the CRISPR-Cas9 system and then outline the current applications of this system as a genome-editing tool in mice and other species, to better model and study human diseases. We also discuss the practical and potential uses of the CRISPR-Cas9 system in kidney research and highlight the further applications of this technology beyond genome editing. Undoubtedly, the CRISPR-Cas9 system holds enormous potential for revolutionizing and accelerating kidney research and therapeutic applications in the future. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Pea early-browning virus -mediated genome editing via the CRISPR/Cas9 system in Nicotiana benthamiana and Arabidopsis

KAUST Repository

Ali, Zahir; Eid, Ayman; Ali, Shawkat; Mahfouz, Magdy M.

2017-01-01

The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system has enabled efficient genome engineering in diverse plant species. However, delivery of genome engineering reagents, such as the single guide RNA (sgRNA), into plant cells remains challenging. Here, we report the engineering of Tobacco rattle virus (TRV) and Pea early browning virus (PEBV) to deliver one or multiple sgRNAs into Nicotiana benthamiana and Arabidopsis thaliana (Col-0) plants that overexpress a nuclear localization signal containing Cas9. Our data showed that TRV and PEBV can deliver sgRNAs into inoculated and systemic leaves, and this resulted in mutagenesis of the targeted genomic loci. Moreover, in N. benthamiana, PEBV-based sgRNA delivery resulted in more targeted mutations than TRV-based delivery. Our data indicate that TRV and PEBV can facilitate plant genome engineering and can be used to produce targeted mutations for functional analysis and other biotechnological applications across diverse plant species.Key message: Delivery of genome engineering reagents into plant cells is challenging and inefficient and this limit the applications of this technology in many plant species. RNA viruses such as TRV and PEBV provide an efficient tool to systemically deliver sgRNAs for targeted genome modification.

Comparative genomic characterization of three Streptococcus parauberis strains in fish pathogen, as assessed by wide-genome analyses.

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Seong-Won Nho

Full Text Available Streptococcus parauberis, which is the main causative agent of streptococcosis among olive flounder (Paralichthys olivaceus in northeast Asia, can be distinctly divided into two groups (type I and type II by an agglutination test. Here, the whole genome sequences of two Japanese strains (KRS-02083 and KRS-02109 were determined and compared with the previously determined genome of a Korean strain (KCTC 11537. The genomes of S. parauberis are intermediate in size and have lower GC contents than those of other streptococci. We annotated 2,236 and 2,048 genes in KRS-02083 and KRS-02109, respectively. Our results revealed that the three S. parauberis strains contain different genomic insertions and deletions. In particular, the genomes of Korean and Japanese strains encode different factors for sugar utilization; the former encodes the phosphotransferase system (PTS for sorbose, whereas the latter encodes proteins for lactose hydrolysis, respectively. And the KRS-02109 strain, specifically, was the type II strain found to be able to resist phage infection through the clustered regularly interspaced short palindromic repeats (CRISPR/Cas system and which might contribute valuably to serologically distribution. Thus, our genome-wide association study shows that polymorphisms can affect pathogen responses, providing insight into biological/biochemical pathways and phylogenetic diversity.

Genome Editing by CRISPR/Cas9: A Game Change in the Genetic Manipulation of Protists.

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Lander, Noelia; Chiurillo, Miguel A; Docampo, Roberto

2016-09-01

Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system has been transformative in biology. Originally discovered as an adaptive prokaryotic immune system, CRISPR/Cas9 has been repurposed for genome editing in a broad range of model organisms, from yeast to mammalian cells. Protist parasites are unicellular organisms producing important human diseases that affect millions of people around the world. For many of these diseases, such as malaria, Chagas disease, leishmaniasis and cryptosporidiosis, there are no effective treatments or vaccines available. The recent adaptation of the CRISPR/Cas9 technology to several protist models will be playing a key role in the functional study of their proteins, in the characterization of their metabolic pathways, and in the understanding of their biology, and will facilitate the search for new chemotherapeutic targets. In this work we review recent studies where the CRISPR/Cas9 system was adapted to protist parasites, particularly to Apicomplexans and trypanosomatids, emphasizing the different molecular strategies used for genome editing of each organism, as well as their advantages. We also discuss the potential usefulness of this technology in the green alga Chlamydomonas reinhardtii. © 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.

Crystallization and preliminary X-ray diffraction analysis of the CRISPR-Cas RNA-silencing Cmr complex.

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Osawa, Takuo; Inanaga, Hideko; Numata, Tomoyuki

2015-06-01

Clustered regularly interspaced short palindromic repeat (CRISPR)-derived RNA (crRNA) and CRISPR-associated (Cas) proteins constitute a prokaryotic adaptive immune system (CRISPR-Cas system) that targets and degrades invading genetic elements. The type III-B CRISPR-Cas Cmr complex, composed of the six Cas proteins (Cmr1-Cmr6) and a crRNA, captures and cleaves RNA complementary to the crRNA guide sequence. Here, a Cmr1-deficient functional Cmr (CmrΔ1) complex composed of Pyrococcus furiosus Cmr2-Cmr3, Archaeoglobus fulgidus Cmr4-Cmr5-Cmr6 and the 39-mer P. furiosus 7.01-crRNA was prepared. The CmrΔ1 complex was cocrystallized with single-stranded DNA (ssDNA) complementary to the crRNA guide by the vapour-diffusion method. The crystals diffracted to 2.1 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the triclinic space group P1, with unit-cell parameters a = 75.5, b = 76.2, c = 139.2 Å, α = 90.3, β = 104.8, γ = 118.6°. The asymmetric unit of the crystals is expected to contain one CmrΔ1-ssDNA complex, with a Matthews coefficient of 2.03 Å(3) Da(-1) and a solvent content of 39.5%.

Deletion of Pr130 Interrupts Cardiac Development in Zebrafish

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Jie Yang

2016-11-01

Full Text Available Protein phosphatase 2 regulatory subunit B, alpha (PPP2R3A, a regulatory subunit of protein phosphatase 2A (PP2A, is a major serine/threonine phosphatase that regulates crucial function in development and growth. Previous research has implied that PPP2R3A was involved in heart failure, and PR130, the largest transcription of PPP2R3A, functioning in the calcium release of sarcoplasmic reticulum (SR, plays an important role in the excitation-contraction (EC coupling. To obtain a better understanding of PR130 functions in myocardium and cardiac development, two pr130-deletion zebrafish lines were generated using clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR-associated proteins (Cas system. Pr130-knockout zebrafish exhibited cardiac looping defects and decreased cardiac function (decreased fractional area and fractional shortening. Hematoxylin and eosin (H&E staining demonstrated reduced cardiomyocytes. Subsequent transmission electron microscopy revealed that the bright and dark bands were narrowed and blurred, the Z- and M-lines were fogged, and the gaps between longitudinal myocardial fibers were increased. Additionally, increased apoptosis was observed in cardiomyocyte in pr130-knockout zebrafish compared to wild-type (WT. Taken together, our results suggest that pr130 is required for normal myocardium formation and efficient cardiac contractile function.

Enhancement of single guide RNA transcription for efficient CRISPR/Cas-based genomic engineering.

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Ui-Tei, Kumiko; Maruyama, Shohei; Nakano, Yuko

2017-06-01

Genomic engineering using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein is a promising approach for targeting the genomic DNA of virtually any organism in a sequence-specific manner. Recent remarkable advances in CRISPR/Cas technology have made it a feasible system for use in therapeutic applications and biotechnology. In the CRISPR/Cas system, a guide RNA (gRNA), interacting with the Cas protein, recognizes a genomic region with sequence complementarity, and the double-stranded DNA at the target site is cleaved by the Cas protein. A widely used gRNA is an RNA polymerase III (pol III)-driven single gRNA (sgRNA), which is produced by artificial fusion of CRISPR RNA (crRNA) and trans-activation crRNA (tracrRNA). However, we identified a TTTT stretch, known as a termination signal of RNA pol III, in the scaffold region of the sgRNA. Here, we revealed that sgRNA carrying a TTTT stretch reduces the efficiency of sgRNA transcription due to premature transcriptional termination, and decreases the efficiency of genome editing. Unexpectedly, it was also shown that the premature terminated sgRNA may have an adverse effect of inducing RNA interference. Such disadvantageous effects were avoided by substituting one base in the TTTT stretch.

Homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae.

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Bao, Zehua; Xiao, Han; Liang, Jing; Zhang, Lu; Xiong, Xiong; Sun, Ning; Si, Tong; Zhao, Huimin

2015-05-15

One-step multiple gene disruption in the model organism Saccharomyces cerevisiae is a highly useful tool for both basic and applied research, but it remains a challenge. Here, we report a rapid, efficient, and potentially scalable strategy based on the type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated proteins (Cas) system to generate multiple gene disruptions simultaneously in S. cerevisiae. A 100 bp dsDNA mutagenizing homologous recombination donor is inserted between two direct repeats for each target gene in a CRISPR array consisting of multiple donor and guide sequence pairs. An ultrahigh copy number plasmid carrying iCas9, a variant of wild-type Cas9, trans-encoded RNA (tracrRNA), and a homology-integrated crRNA cassette is designed to greatly increase the gene disruption efficiency. As proof of concept, three genes, CAN1, ADE2, and LYP1, were simultaneously disrupted in 4 days with an efficiency ranging from 27 to 87%. Another three genes involved in an artificial hydrocortisone biosynthetic pathway, ATF2, GCY1, and YPR1, were simultaneously disrupted in 6 days with 100% efficiency. This homology-integrated CRISPR (HI-CRISPR) strategy represents a powerful tool for creating yeast strains with multiple gene knockouts.

Occurrence and activity of a type II CRISPR-Cas system in Lactobacillus gasseri.

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Sanozky-Dawes, Rosemary; Selle, Kurt; O'Flaherty, Sarah; Klaenhammer, Todd; Barrangou, Rodolphe

2015-09-01

Bacteria encode clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated genes (cas), which collectively form an RNA-guided adaptive immune system against invasive genetic elements. In silico surveys have revealed that lactic acid bacteria harbour a prolific and diverse set of CRISPR-Cas systems. Thus, the natural evolutionary role of CRISPR-Cas systems may be investigated in these ecologically, industrially, scientifically and medically important microbes. In this study, 17 Lactobacillus gasseri strains were investigated and 6 harboured a type II-A CRISPR-Cas system, with considerable diversity in array size and spacer content. Several of the spacers showed similarity to phage and plasmid sequences, which are typical targets of CRISPR-Cas immune systems. Aligning the protospacers facilitated inference of the protospacer adjacent motif sequence, determined to be 5'-NTAA-3' flanking the 3' end of the protospacer. The system in L. gasseri JV-V03 and NCK 1342 interfered with transforming plasmids containing sequences matching the most recently acquired CRISPR spacers in each strain. We report the distribution and function of a native type II-A CRISPR-Cas system in the commensal species L. gasseri. Collectively, these results open avenues for applications for bacteriophage protection and genome modification in L. gasseri, and contribute to the fundamental understanding of CRISPR-Cas systems in bacteria.

A CRISPR-Cas system enhances envelope integrity mediating antibiotic resistance and inflammasome evasion.

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Sampson, Timothy R; Napier, Brooke A; Schroeder, Max R; Louwen, Rogier; Zhao, Jinshi; Chin, Chui-Yoke; Ratner, Hannah K; Llewellyn, Anna C; Jones, Crystal L; Laroui, Hamed; Merlin, Didier; Zhou, Pei; Endtz, Hubert P; Weiss, David S

2014-07-29

Clustered, regularly interspaced, short palindromic repeats-CRISPR associated (CRISPR-Cas) systems defend bacteria against foreign nucleic acids, such as during bacteriophage infection and transformation, processes which cause envelope stress. It is unclear if these machineries enhance membrane integrity to combat this stress. Here, we show that the Cas9-dependent CRISPR-Cas system of the intracellular bacterial pathogen Francisella novicida is involved in enhancing envelope integrity through the regulation of a bacterial lipoprotein. This action ultimately provides increased resistance to numerous membrane stressors, including antibiotics. We further find that this previously unappreciated function of Cas9 is critical during infection, as it promotes evasion of the host innate immune absent in melanoma 2/apoptosis associated speck-like protein containing a CARD (AIM2/ASC) inflammasome. Interestingly, the attenuation of the cas9 mutant is complemented only in mice lacking both the AIM2/ASC inflammasome and the bacterial lipoprotein sensor Toll-like receptor 2, but not in single knockout mice, demonstrating that Cas9 is essential for evasion of both pathways. These data represent a paradigm shift in our understanding of the function of CRISPR-Cas systems as regulators of bacterial physiology and provide a framework with which to investigate the roles of these systems in myriad bacteria, including pathogens and commensals.

Melatonin Promotes the In Vitro Development of Microinjected Pronuclear Mouse Embryos via Its Anti-Oxidative and Anti-Apoptotic Effects.

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Tian, Xiuzhi; Wang, Feng; Zhang, Lu; Ji, Pengyun; Wang, Jing; Lv, Dongying; Li, Guangdong; Chai, Menglong; Lian, Zhengxing; Liu, Guoshi

2017-05-05

CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats) combined with pronuclear microinjection has become the most effective method for producing transgenic animals. However, the relatively low embryo developmental rate limits its application. In the current study, it was observed that 10 -7 M melatonin is considered an optimum concentration and significantly promoted the in vitro development of murine microinjected pronuclear embryos, as indicated by the increased blastocyst rate, hatching blastocyst rate and blastocyst cell number. When these blastocysts were implanted into recipient mice, the pregnancy rate and birth rate were significantly higher than those of the microinjected control, respectively. Mechanistic studies revealed that melatonin treatment reduced reactive oxygen species (ROS) production and cellular apoptosis during in vitro embryo development and improved the quality of the blastocysts. The implantation of quality-improved blastocysts led to elevated pregnancy and birth rates. In conclusion, the results revealed that the anti-oxidative and anti-apoptotic activities of melatonin improved the quality of microinjected pronuclear embryos and subsequently increased both the efficiency of embryo implantation and the birth rate of the pups. Therefore, the melatonin supplementation may provide a novel alternative method for generating large numbers of transgenic mice and this method can probably be used in human-assisted reproduction and genome editing.

Host specific diversity in Lactobacillus johnsonii as evidenced by a major chromosomal inversion and phage resistance mechanisms.

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Guinane, Caitriona M; Kent, Robert M; Norberg, Sarah; Hill, Colin; Fitzgerald, Gerald F; Stanton, Catherine; Ross, R Paul

2011-04-20

Genetic diversity and genomic rearrangements are a driving force in bacterial evolution and niche adaptation. We sequenced and annotated the genome of Lactobacillus johnsonii DPC6026, a strain isolated from the porcine intestinal tract. Although the genome of DPC6026 is similar in size (1.97 mbp) and GC content (34.8%) to the sequenced human isolate L. johnsonii NCC 533, a large symmetrical inversion of approximately 750 kb differentiated the two strains. Comparative analysis among 12 other strains of L. johnsonii including 8 porcine, 3 human and 1 poultry isolate indicated that the genome architecture found in DPC6026 is more common within the species than that of NCC 533. Furthermore a number of unique features were annotated in DPC6026, some of which are likely to have been acquired by horizontal gene transfer (HGT) and contribute to protection against phage infection. A putative type III restriction-modification system was identified, as were novel Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) elements. Interestingly, these particular elements are not widely distributed among L. johnsonii strains. Taken together these data suggest intra-species genomic rearrangements and significant genetic diversity within the L. johnsonii species and indicate towards a host-specific divergence of L. johnsonii strains with respect to genome inversion and phage exposure.

Host specific diversity in Lactobacillus johnsonii as evidenced by a major chromosomal inversion and phage resistance mechanisms.

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Caitriona M Guinane

Full Text Available Genetic diversity and genomic rearrangements are a driving force in bacterial evolution and niche adaptation. We sequenced and annotated the genome of Lactobacillus johnsonii DPC6026, a strain isolated from the porcine intestinal tract. Although the genome of DPC6026 is similar in size (1.97 mbp and GC content (34.8% to the sequenced human isolate L. johnsonii NCC 533, a large symmetrical inversion of approximately 750 kb differentiated the two strains. Comparative analysis among 12 other strains of L. johnsonii including 8 porcine, 3 human and 1 poultry isolate indicated that the genome architecture found in DPC6026 is more common within the species than that of NCC 533. Furthermore a number of unique features were annotated in DPC6026, some of which are likely to have been acquired by horizontal gene transfer (HGT and contribute to protection against phage infection. A putative type III restriction-modification system was identified, as were novel Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR elements. Interestingly, these particular elements are not widely distributed among L. johnsonii strains. Taken together these data suggest intra-species genomic rearrangements and significant genetic diversity within the L. johnsonii species and indicate towards a host-specific divergence of L. johnsonii strains with respect to genome inversion and phage exposure.

Heritable genome editing with CRISPR/Cas9 in the silkworm, Bombyx mori.

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Wei Wei

Full Text Available We report the establishment of an efficient and heritable gene mutagenesis method in the silkworm Bombyx mori using modified type II clustered regularly interspaced short palindromic repeats (CRISPR with an associated protein (Cas9 system. Using four loci Bm-ok, BmKMO, BmTH, and Bmtan as candidates, we proved that genome alterations at specific sites could be induced by direct microinjection of specific guide RNA and Cas9-mRNA into silkworm embryos. Mutation frequencies of 16.7-35.0% were observed in the injected generation, and DNA fragments deletions were also noted. Bm-ok mosaic mutants were used to test for mutant heritability due to the easily determined translucent epidermal phenotype of Bm-ok-disrupted cells. Two crossing strategies were used. In the first, injected Bm-ok moths were crossed with wild-type moths, and a 28.6% frequency of germline mutation transmission was observed. In the second strategy, two Bm-ok mosaic mutant moths were crossed with each other, and 93.6% of the offsprings appeared mutations in both alleles of Bm-ok gene (compound heterozygous. In summary, the CRISPR/Cas9 system can act as a highly specific and heritable gene-editing tool in Bombyx mori.

Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers

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Gao, Xuefei; Tsang, Jason C.H.; Gaba, Fortis; Wu, Donghai; Lu, Liming; Liu, Pentao

2014-01-01

The transcription activator–like effectors (TALEs) and the RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR) associated protein (Cas9) utlilize distinct molecular mechanisms in targeting site recognition. The two proteins can be modified to carry additional functional domains to regulate expression of genomic loci in mammalian cells. In this study, we have compared the two systems in activation and suppression of the Oct4 and Nanog loci by targeting their enhancers. Although both are able to efficiently activate the luciferase reporters, the CRISPR/dCas9 system is much less potent in activating the endogenous loci and in the application of reprogramming somatic cells to iPS cells. Nevertheless, repression by CRISPR/dCas9 is comparable to or even better than TALE repressors. We demonstrated that dCas9 protein binding results in significant physical interference to binding of native transcription factors at enhancer, less efficient active histone markers induction or recruitment of activating complexes in gene activation. This study thus highlighted the merits and drawbacks of transcription regulation by each system. A combined approach of TALEs and CRISPR/dCas9 should provide an optimized solution to regulate genomic loci and to study genetic elements such as enhancers in biological processes including somatic cell reprogramming and guided differentiation. PMID:25223790

Tackling HIV Persistence: Pharmacological versus CRISPR-Based Shock Strategies.

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Darcis, Gilles; Das, Atze T; Berkhout, Ben

2018-03-29

Jan Svoboda studied aspects of viral latency, in particular with respect to disease induction by avian RNA tumor viruses, which were later renamed as part of the extended retrovirus family. The course of retroviral pathogenesis is intrinsically linked to their unique property of integrating the DNA copy of the retroviral genome into that of the host cell, thus forming the provirus. Retroviral latency has recently become of major clinical interest to allow a better understanding of why we can effectively block the human immunodeficiency virus type 1 (HIV-1) in infected individuals with antiviral drugs, yet never reach a cure. We will discuss HIV-1 latency and its direct consequence-the formation of long-lasting HIV-1 reservoirs. We next focus on one of the most explored strategies in tackling HIV-1 reservoirs-the "shock and kill" strategy-which describes the broadly explored pharmacological way of kicking the latent provirus, with subsequent killing of the virus-producing cell by the immune system. We furthermore present how the clustered regularly interspaced palindromic repeats (CRISPR) and associated protein (Cas) system can be harnessed to reach the same objective by reactivating HIV-1 gene expression from latency. We will review the benefits and drawbacks of these different cure strategies.

Gene Therapy for Chronic HBV-Can We Eliminate cccDNA?

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Bloom, Kristie; Maepa, Mohube Betty; Ely, Abdullah; Arbuthnot, Patrick

2018-04-12

Chronic infection with the hepatitis B virus (HBV) is a global health concern and accounts for approximately 1 million deaths annually. Amongst other limitations of current anti-HBV treatment, failure to eliminate the viral covalently closed circular DNA (cccDNA) and emergence of resistance remain the most worrisome. Viral rebound from latent episomal cccDNA reservoirs occurs following cessation of therapy, patient non-compliance, or the development of escape mutants. Simultaneous viral co-infections, such as by HIV-1, further complicate therapeutic interventions. These challenges have prompted development of novel targeted hepatitis B therapies. Given the ease with which highly specific and potent nucleic acid therapeutics can be rationally designed, gene therapy has generated interest for antiviral application. Gene therapy strategies developed for HBV include gene silencing by harnessing RNA interference, transcriptional inhibition through epigenetic modification of target DNA, genome editing by designer nucleases, and immune modulation with cytokines. DNA-binding domains and effectors based on the zinc finger (ZF), transcription activator-like effector (TALE), and clustered regularly interspaced short palindromic repeat (CRISPR) systems are remarkably well suited to targeting episomal cccDNA. This review discusses recent developments and challenges facing the field of anti-HBV gene therapy, its potential curative significance and the progress towards clinical application.

Pea early-browning virus -mediated genome editing via the CRISPR/Cas9 system in Nicotiana benthamiana and Arabidopsis

KAUST Repository

Ali, Zahir

2017-10-17

The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system has enabled efficient genome engineering in diverse plant species. However, delivery of genome engineering reagents, such as the single guide RNA (sgRNA), into plant cells remains challenging. Here, we report the engineering of Tobacco rattle virus (TRV) and Pea early browning virus (PEBV) to deliver one or multiple sgRNAs into Nicotiana benthamiana and Arabidopsis thaliana (Col-0) plants that overexpress a nuclear localization signal containing Cas9. Our data showed that TRV and PEBV can deliver sgRNAs into inoculated and systemic leaves, and this resulted in mutagenesis of the targeted genomic loci. Moreover, in N. benthamiana, PEBV-based sgRNA delivery resulted in more targeted mutations than TRV-based delivery. Our data indicate that TRV and PEBV can facilitate plant genome engineering and can be used to produce targeted mutations for functional analysis and other biotechnological applications across diverse plant species.Key message: Delivery of genome engineering reagents into plant cells is challenging and inefficient and this limit the applications of this technology in many plant species. RNA viruses such as TRV and PEBV provide an efficient tool to systemically deliver sgRNAs for targeted genome modification.

Genetic Basis of Melanin Pigmentation in Butterfly Wings.

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Zhang, Linlin; Martin, Arnaud; Perry, Michael W; van der Burg, Karin R L; Matsuoka, Yuji; Monteiro, Antónia; Reed, Robert D

2017-04-01

Despite the variety, prominence, and adaptive significance of butterfly wing patterns, surprisingly little is known about the genetic basis of wing color diversity. Even though there is intense interest in wing pattern evolution and development, the technical challenge of genetically manipulating butterflies has slowed efforts to functionally characterize color pattern development genes. To identify candidate wing pigmentation genes, we used RNA sequencing to characterize transcription across multiple stages of butterfly wing development, and between different color pattern elements, in the painted lady butterfly Vanessa cardui This allowed us to pinpoint genes specifically associated with red and black pigment patterns. To test the functions of a subset of genes associated with presumptive melanin pigmentation, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing in four different butterfly genera. pale , Ddc , and yellow knockouts displayed reduction of melanin pigmentation, consistent with previous findings in other insects. Interestingly, however, yellow-d , ebony , and black knockouts revealed that these genes have localized effects on tuning the color of red, brown, and ochre pattern elements. These results point to previously undescribed mechanisms for modulating the color of specific wing pattern elements in butterflies, and provide an expanded portrait of the insect melanin pathway. Copyright © 2017 by the Genetics Society of America.

CRISPR Typing and Antibiotic Resistance Correlates with Polyphyletic Distribution in Human Isolates of Salmonella Kentucky.

Science.gov (United States)

Vosik, Dorothy; Tewari, Deepanker; Dettinger, Lisa; M'ikanatha, Nkuchia M; Shariat, Nikki W

2018-02-01

Although infrequently associated with reported salmonellosis in humans, Salmonella enterica, subsp. enterica serovar Kentucky (ser. Kentucky) is the most common nonclinical, nonhuman serovar reported in the United States. The goal of this study was to use Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-multi-virulence-locus sequence typing (MVLST) to subtype a collection of human clinical isolates of ser. Kentucky submitted to the Pennsylvania Department of Health and to determine the extent of antibiotic resistance in these strains. This analysis highlighted the polyphyletic nature of ser. Kentucky, and separated our isolates into two groups, Group I and Group II, which were equally represented in our collection. Furthermore, antimicrobial susceptibility testing on all isolates using a National Antimicrobial Resistance Monitoring System (NARMS) panel of antibiotics demonstrated that resistance profiles could be divided into two groups. Group I isolates were resistant to cephems and penicillins, whereas Group II isolates were resistant to quinolones, gentamicin, and sulfisoxazole. Collectively, 50% of isolates were resistant to three or more classes of antibiotics and 30% were resistant to five or more classes. The correlation of antibiotic resistance with the two different lineages may reflect adaptation within two distinct reservoirs of ser. Kentucky, with differential exposure to antimicrobials.

Engineered Viruses as Genome Editing Devices

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Chen, Xiaoyu; Gonçalves, Manuel A F V

2016-01-01

Genome editing based on sequence-specific designer nucleases, also known as programmable nucleases, seeks to modify in a targeted and precise manner the genetic information content of living cells. Delivering into cells designer nucleases alone or together with donor DNA templates, which serve as surrogate homologous recombination (HR) substrates, can result in gene knockouts or gene knock-ins, respectively. As engineered replication-defective viruses, viral vectors are having an increasingly important role as delivery vehicles for donor DNA templates and designer nucleases, namely, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated Cas9 (CRISPR−Cas9) nucleases, also known as RNA-guided nucleases (RGNs). We review this dual role played by engineered viral particles on genome editing while focusing on their main scaffolds, consisting of lentiviruses, adeno-associated viruses, and adenoviruses. In addition, the coverage of the growing body of research on the repurposing of viral vectors as delivery systems for genome editing tools is complemented with information regarding their main characteristics, pros, and cons. Finally, this information is framed by a concise description of the chief principles, tools, and applications of the genome editing field as a whole. PMID:26336974

Crystallization and preliminary X-ray diffraction analysis of the Cmr2–Cmr3 subcomplex in the CRISPR–Cas RNA-silencing effector complex

Energy Technology Data Exchange (ETDEWEB)

Osawa, Takuo; Inanaga, Hideko; Numata, Tomoyuki [National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba-shi, Ibaraki 305-8566 (Japan)

2013-04-30

The Cmr2–Cmr3 subcomplex from P. furiosus was co-crystallized with 3′-AMP. X-ray diffraction data for the crystals were collected to 2.6 Å resolution using a synchrotron-radiation source. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci, found in prokaryotes, are transcribed to produce CRISPR RNAs (crRNAs). The Cmr proteins (Cmr1–6) and crRNA form a ribonucleoprotein complex that degrades target RNAs derived from invading genetic elements. Cmr2dHD, a Cmr2 variant lacking the N-terminal putative HD nuclease domain, and Cmr3 were co-expressed in Escherichia coli cells and co-purified as a complex. The Cmr2dHD–Cmr3 complex was co-crystallized with 3′-AMP by the vapour-diffusion method. The crystals diffracted to 2.6 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the orthorhombic space group I222, with unit-cell parameters a = 103.9, b = 136.7, c = 192.0 Å. The asymmetric unit of the crystals is expected to contain one Cmr2dHD–Cmr3 complex with a Matthews coefficient of 3.0 Å{sup 3} Da{sup −1} and a solvent content of 59%.

CRISPR/Cas-mediated targeted mutagenesis in Daphnia magna.

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Takashi Nakanishi

Full Text Available The water flea Daphnia magna has been used as an animal model in ecology, evolution, and environmental sciences. Thanks to the recent progress in Daphnia genomics, genetic information such as the draft genome sequence and expressed sequence tags (ESTs is now available. To investigate the relationship between phenotypes and the available genetic information about Daphnia, some gene manipulation methods have been developed. However, a technique to induce targeted mutagenesis into Daphnia genome remains elusive. To overcome this problem, we focused on an emerging genome editing technique mediated by the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas system to introduce genomic mutations. In this study, we targeted a functionally conserved regulator of eye development, the eyeless gene in D. magna. When we injected Cas9 mRNAs and eyeless-targeting guide RNAs into eggs, 18-47% of the survived juveniles exhibited abnormal eye morphology. After maturation, up to 8.2% of the adults produced progenies with deformed eyes, which carried mutations in the eyeless loci. These results showed that CRISPR/Cas system could introduce heritable mutations into the endogenous eyeless gene in D. magna. This is the first report of a targeted gene knockout technique in Daphnia and will be useful in uncovering Daphnia gene functions.

Application of the nanobiotechnology with the system CRISP-Cas

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Liceth Xiomara Sáenz-Castiblanco

2017-12-01

Full Text Available Introduction: Nanobiotechnology and synthetic biology are sciences that impact today with the launching of innovative and beneficial applications for the human being. These sciences have been amalgamated to manufacture new components for the construction of totally artificial cells and the creation of synthetic biomolecules. Objective: To know the applications of nanobiotechnology related to the use of the system CRISPR/Cas in the storage of bacterial DNA and therapeutic alternatives. Materials and methods: A bibliographical review on the main applications of nanobiotechnology was carried out in ScienceDirect, SciELO, PubMed databases and in magazines such as: Nature Biotechnology, Biochemistry, Science and Journal Microbiology. Results: The literature review describes and analyzes the new nanobiotechnology applications used to write information in the genetic code of bacterial cells, in which the system is used based on short grouped and regularly interspaced palindromic repetitions (CRISPR/Cas and the production of synthetic DNA, as well as therapeutic alternatives related to gene therapy. Conclusion: Among the nanobiotechnology applications, two methods to record information in the DNA of bacterial cells Escherichia coli and Sulfolobus Tokodai have been shown, which are linked to the use of the system CRISPR/Cas and the production of synthetic DNA, as well as the use of CRISPR/Cas in gene and cellular therapy.

Integrative Analysis of CRISPR/Cas9 Target Sites in the Human HBB Gene

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Yumei Luo

2015-01-01

Full Text Available Recently, the clustered regularly interspaced short palindromic repeats (CRISPR system has emerged as a powerful customizable artificial nuclease to facilitate precise genetic correction for tissue regeneration and isogenic disease modeling. However, previous studies reported substantial off-target activities of CRISPR system in human cells, and the enormous putative off-target sites are labor-intensive to be validated experimentally, thus motivating bioinformatics methods for rational design of CRISPR system and prediction of its potential off-target effects. Here, we describe an integrative analytical process to identify specific CRISPR target sites in the human β-globin gene (HBB and predict their off-target effects. Our method includes off-target analysis in both coding and noncoding regions, which was neglected by previous studies. It was found that the CRISPR target sites in the introns have fewer off-target sites in the coding regions than those in the exons. Remarkably, target sites containing certain transcriptional factor motif have enriched binding sites of relevant transcriptional factor in their off-target sets. We also found that the intron sites have fewer SNPs, which leads to less variation of CRISPR efficiency in different individuals during clinical applications. Our studies provide a standard analytical procedure to select specific CRISPR targets for genetic correction.

Gene Disruption Technologies Have the Potential to Transform Stored Product Insect Pest Control

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Lindsey C. Perkin

2016-09-01

Full Text Available Stored product insects feed on grains and processed commodities manufactured from grain post-harvest, reducing the nutritional value and contaminating food. Currently, the main defense against stored product insect pests is the pesticide fumigant phosphine. Phosphine is highly toxic to all animals, but is the most effective and economical control method, and thus is used extensively worldwide. However, many insect populations have become resistant to phosphine, in some cases to very high levels. New, environmentally benign and more effective control strategies are needed for stored product pests. RNA interference (RNAi may overcome pesticide resistance by targeting the expression of genes that contribute to resistance in insects. Most data on RNAi in stored product insects is from the coleopteran genetic model, Tribolium castaneum, since it has a strong RNAi response via injection of double stranded RNA (dsRNA in any life stage. Additionally, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR technology has been suggested as a potential resource for new pest control strategies. In this review we discuss background information on both gene disruption technologies and summarize the advances made in terms of molecular pest management in stored product insects, mainly T. castaneum, as well as complications and future needs.

RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors

KAUST Repository

Piatek, Agnieszka Anna; Ali, Zahir; Baazim, Hatoon; Li, Lixin; Abulfaraj, Aala A.; Alshareef, Sahar; Aouida, Mustapha; Mahfouz, Magdy M.

2014-01-01

Targeted genomic regulation is a powerful approach to accelerate trait discovery and development in agricultural biotechnology. Bacteria and archaea use clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) regulatory systems for adaptive molecular immunity against foreign nucleic acids introduced by invading phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing in many cell types and organisms. A recent study used the catalytically inactive Cas9 (dCas9) protein combined with guide-RNAs (gRNAs) as a DNA-targeting platform to modulate gene expression in bacterial, yeast, and human cells. Here, we modified this DNA-targeting platform for targeted transcriptional regulation in planta by developing chimeric dCas9-based transcriptional activators and repressors. To generate transcriptional activators, we fused the dCas9 C-terminus with the activation domains of EDLL and TAL effectors. To generate a transcriptional repressor, we fused the dCas9 C-terminus with the SRDX repression domain. Our data demonstrate that dCas9 fusion with the EDLL activation domain (dCas9:EDLL) and the TAL activation domain (dCas9:TAD), guided by gRNAs complementary to selected promoter elements, induce strong transcriptional activation on Bs3

CCR5 Targeted Cell Therapy for HIV and Prevention of Viral Escape

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Gero Hütter

2015-07-01

Full Text Available Allogeneic transplantation with CCR5-delta 32 (CCR5-d32 homozygous stem cells in an HIV infected individual in 2008, led to a sustained virus control and probably eradication of HIV. Since then there has been a high degree of interest to translate this approach to a wider population. There are two cellular ways to do this. The first one is to use a CCR5 negative cell source e.g., hematopoietic stem cells (HSC to copy the initial finding. However, a recent case of a second allogeneic transplantation with CCR5-d32 homozygous stem cells suffered from viral escape of CXCR4 quasi-species. The second way is to knock down CCR5 expression by gene therapy. Currently, there are five promising techniques, three of which are presently being tested clinically. These techniques include zinc finger nucleases (ZFN, clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 nuclease (CRISPR/Cas9, transcription activator-like effectors nuclease (TALEN, short hairpin RNA (shRNA, and a ribozyme. While there are multiple gene therapy strategies being tested, in this review we reflect on our current knowledge of inhibition of CCR5 specifically and whether this approach allows for consequent viral escape.

CRISPR Detection From Short Reads Using Partial Overlap Graphs.

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Ben-Bassat, Ilan; Chor, Benny

2016-06-01

Clustered regularly interspaced short palindromic repeats (CRISPR) are structured regions in bacterial and archaeal genomes, which are part of an adaptive immune system against phages. CRISPRs are important for many microbial studies and are playing an essential role in current gene editing techniques. As such, they attract substantial research interest. The exponential growth in the amount of bacterial sequence data in recent years enables the exploration of CRISPR loci in more and more species. Most of the automated tools that detect CRISPR loci rely on fully assembled genomes. However, many assemblers do not handle repetitive regions successfully. The first tool to work directly on raw sequence data is Crass, which requires reads that are long enough to contain two copies of the same repeat. We present a method to identify CRISPR repeats from raw sequence data of short reads. The algorithm is based on an observation differentiating CRISPR repeats from other types of repeats, and it involves a series of partial constructions of the overlap graph. This enables us to avoid many of the difficulties that assemblers face, as we merely aim to identify the repeats that belong to CRISPR loci. A preliminary implementation of the algorithm shows good results and detects CRISPR repeats in cases where other existing tools fail to do so.

Crystallization and preliminary X-ray diffraction analysis of the Cmr2–Cmr3 subcomplex in the CRISPR–Cas RNA-silencing effector complex

International Nuclear Information System (INIS)

Osawa, Takuo; Inanaga, Hideko; Numata, Tomoyuki

2013-01-01

The Cmr2–Cmr3 subcomplex from P. furiosus was co-crystallized with 3′-AMP. X-ray diffraction data for the crystals were collected to 2.6 Å resolution using a synchrotron-radiation source. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci, found in prokaryotes, are transcribed to produce CRISPR RNAs (crRNAs). The Cmr proteins (Cmr1–6) and crRNA form a ribonucleoprotein complex that degrades target RNAs derived from invading genetic elements. Cmr2dHD, a Cmr2 variant lacking the N-terminal putative HD nuclease domain, and Cmr3 were co-expressed in Escherichia coli cells and co-purified as a complex. The Cmr2dHD–Cmr3 complex was co-crystallized with 3′-AMP by the vapour-diffusion method. The crystals diffracted to 2.6 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the orthorhombic space group I222, with unit-cell parameters a = 103.9, b = 136.7, c = 192.0 Å. The asymmetric unit of the crystals is expected to contain one Cmr2dHD–Cmr3 complex with a Matthews coefficient of 3.0 Å 3 Da −1 and a solvent content of 59%

The Role of CRISPR-Cas Systems in Virulence of Pathogenic Bacteria

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Staals, Raymond H. J.; Endtz, Hubert P.; van Baarlen, Peter; van der Oost, John

2014-01-01

SUMMARY Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are present in many bacterial and archaeal genomes. Since the discovery of the typical CRISPR loci in the 1980s, well before their physiological role was revealed, their variable sequences have been used as a complementary typing tool in diagnostic, epidemiologic, and evolutionary analyses of prokaryotic strains. The discovery that CRISPR spacers are often identical to sequence fragments of mobile genetic elements was a major breakthrough that eventually led to the elucidation of CRISPR-Cas as an adaptive immunity system. Key elements of this unique prokaryotic defense system are small CRISPR RNAs that guide nucleases to complementary target nucleic acids of invading viruses and plasmids, generally followed by the degradation of the invader. In addition, several recent studies have pointed at direct links of CRISPR-Cas to regulation of a range of stress-related phenomena. An interesting example concerns a pathogenic bacterium that possesses a CRISPR-associated ribonucleoprotein complex that may play a dual role in defense and/or virulence. In this review, we describe recently reported cases of potential involvement of CRISPR-Cas systems in bacterial stress responses in general and bacterial virulence in particular. PMID:24600041

Culture time of vitrified/warmed zygotes before microinjection affects the production efficiency of CRISPR-Cas9-mediated knock-in mice.

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Nakagawa, Yoshiko; Sakuma, Tetsushi; Nishimichi, Norihisa; Yokosaki, Yasuyuki; Takeo, Toru; Nakagata, Naomi; Yamamoto, Takashi

2017-05-15

Robust reproductive engineering techniques are required for the efficient and rapid production of genetically modified mice. We have reported the efficient production of genome-edited mice using reproductive engineering techniques, such as ultra-superovulation, in vitro fertilization (IVF) and vitrification/warming of zygotes. We usually use vitrified/warmed fertilized oocytes created by IVF for microinjection because of work efficiency and flexible scheduling. Here, we investigated whether the culture time of zygotes before microinjection influences the efficiency of producing knock-in mice. Knock-in mice were generated using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system and single-stranded oligodeoxynucleotide (ssODN) or PITCh (Precise Integration into Target Chromosome) system, a method of integrating a donor vector assisted by microhomology-mediated end-joining. The cryopreserved fertilized oocytes were warmed, cultured for several hours and microinjected at different timings. Microinjection was performed with Cas9 protein, guide RNA(s), and an ssODN or PITCh donor plasmid for the ssODN knock-in and the PITCh knock-in, respectively. Different production efficiencies of knock-in mice were observed by changing the timing of microinjection. Our study provides useful information for the CRISPR-Cas9-based generation of knock-in mice. © 2017. Published by The Company of Biologists Ltd.

Loss of CIB2 Causes Profound Hearing Loss and Abolishes Mechanoelectrical Transduction in Mice

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Yanfei Wang

2017-12-01

Full Text Available Calcium and integrin-binding protein 2 (CIB2 belongs to a protein family with four known members, CIB1 through CIB4, which are characterized by multiple calcium-binding EF-hand domains. Among the family members, the Cib1 and Cib2 genes are expressed in mouse cochlear hair cells, and mutations in the human CIB2 gene have been associated with nonsyndromic deafness DFNB48 and syndromic deafness USH1J. To further explore the function of CIB1 and CIB2 in hearing, we established Cib1 and Cib2 knockout mice using the clustered regularly interspaced short palindromic repeat (CRISPR-associated Cas9 nuclease (CRISPR/Cas9 genome editing technique. We found that loss of CIB1 protein does not affect auditory function, whereas loss of CIB2 protein causes profound hearing loss in mice. Further investigation revealed that hair cell stereocilia development is affected in Cib2 knockout mice. Noticeably, loss of CIB2 abolishes mechanoelectrical transduction (MET currents in auditory hair cells. In conclusion, we show here that although both CIB1 and CIB2 are readily detected in the cochlea, only loss of CIB2 results in profound hearing loss, and that CIB2 is essential for auditory hair cell MET.

Efficient CRISPR/Cas9-based gene knockout in watermelon.

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Tian, Shouwei; Jiang, Linjian; Gao, Qiang; Zhang, Jie; Zong, Mei; Zhang, Haiying; Ren, Yi; Guo, Shaogui; Gong, Guoyi; Liu, Fan; Xu, Yong

2017-03-01

CRISPR/Cas9 system can precisely edit genomic sequence and effectively create knockout mutations in T0 generation watermelon plants. Genome editing offers great advantage to reveal gene function and generate agronomically important mutations to crops. Recently, RNA-guided genome editing system using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been applied to several plant species, achieving successful targeted mutagenesis. Here, we report the genome of watermelon, an important fruit crop, can also be precisely edited by CRISPR/Cas9 system. ClPDS, phytoene desaturase in watermelon, was selected as the target gene because its mutant bears evident albino phenotype. CRISPR/Cas9 system performed genome editing, such as insertions or deletions at the expected position, in transfected watermelon protoplast cells. More importantly, all transgenic watermelon plants harbored ClPDS mutations and showed clear or mosaic albino phenotype, indicating that CRISPR/Cas9 system has technically 100% of genome editing efficiency in transgenic watermelon lines. Furthermore, there were very likely no off-target mutations, indicated by examining regions that were highly homologous to sgRNA sequences. Our results show that CRISPR/Cas9 system is a powerful tool to effectively create knockout mutations in watermelon.

Coordinate-invariant regularization

International Nuclear Information System (INIS)

Halpern, M.B.

1987-01-01

A general phase-space framework for coordinate-invariant regularization is given. The development is geometric, with all regularization contained in regularized DeWitt Superstructures on field deformations. Parallel development of invariant coordinate-space regularization is obtained by regularized functional integration of the momenta. As representative examples of the general formulation, the regularized general non-linear sigma model and regularized quantum gravity are discussed. copyright 1987 Academic Press, Inc

Functional analysis of SH3 domain containing ring finger 2 during the myogenic differentiation of quail myoblast cells

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Si Won Kim

2017-08-01

Full Text Available Objective Owing to the public availability of complete genome sequences, including avian species, massive bioinformatics analyses may be conducted for computational gene prediction and the identification of gene regulatory networks through various informatics tools. However, to evaluate the biofunctional activity of a predicted target gene, in vivo and in vitro functional genomic analyses should be a prerequisite. Methods Due to a lack of quail genomic sequence information, we first identified the partial genomic structure and sequences of the quail SH3 domain containing ring finger 2 (SH3RF2 gene. Subsequently, SH3RF2 was knocked out using clustered regularly interspaced short palindromic repeat/Cas9 technology and single cell-derived SH3RF2 mutant sublines were established to study the biofunctional activity of SH3RF2 in quail myoblast (QM7 cells during muscle differentiation. Results Through a T7 endonuclease I assay and genotyping analysis, we established an SH3RF2 knockout (KO QM7#4 subline with 61 and 155 nucleotide deletion mutations in SH3RF2. After the induction of myotube differentiation, the expression profiles were analyzed and compared between regular QM7 and SH3RF2 KO QM7#4 cells by global RNA sequencing and bioinformatics analysis. Conclusion We did not detect any statistically significant role of SH3RF2 during myotube differentiation in QM7 myoblast cells. However, additional experiments are necessary to examine the biofunctional activity of SH3RF2 in cell proliferation and muscle growth.

Selection of regularization parameter for l1-regularized damage detection

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Hou, Rongrong; Xia, Yong; Bao, Yuequan; Zhou, Xiaoqing

2018-06-01

The l1 regularization technique has been developed for structural health monitoring and damage detection through employing the sparsity condition of structural damage. The regularization parameter, which controls the trade-off between data fidelity and solution size of the regularization problem, exerts a crucial effect on the solution. However, the l1 regularization problem has no closed-form solution, and the regularization parameter is usually selected by experience. This study proposes two strategies of selecting the regularization parameter for the l1-regularized damage detection problem. The first method utilizes the residual and solution norms of the optimization problem and ensures that they are both small. The other method is based on the discrepancy principle, which requires that the variance of the discrepancy between the calculated and measured responses is close to the variance of the measurement noise. The two methods are applied to a cantilever beam and a three-story frame. A range of the regularization parameter, rather than one single value, can be determined. When the regularization parameter in this range is selected, the damage can be accurately identified even for multiple damage scenarios. This range also indicates the sensitivity degree of the damage identification problem to the regularization parameter.

Improved image quality of cone beam CT scans for radiotherapy image guidance using fiber-interspaced antiscatter grid.

Science.gov (United States)

Stankovic, Uros; van Herk, Marcel; Ploeger, Lennert S; Sonke, Jan-Jakob

2014-06-01

Medical linear accelerator mounted cone beam CT (CBCT) scanner provides useful soft tissue contrast for purposes of image guidance in radiotherapy. The presence of extensive scattered radiation has a negative effect on soft tissue visibility and uniformity of CBCT scans. Antiscatter grids (ASG) are used in the field of diagnostic radiography to mitigate the scatter. They usually do increase the contrast of the scan, but simultaneously increase the noise. Therefore, and considering other scatter mitigation mechanisms present in a CBCT scanner, the applicability of ASGs with aluminum interspacing for a wide range of imaging conditions has been inconclusive in previous studies. In recent years, grids using fiber interspacers have appeared, providing grids with higher scatter rejection while maintaining reasonable transmission of primary radiation. The purpose of this study was to evaluate the impact of one such grid on CBCT image quality. The grid used (Philips Medical Systems) had ratio of 21:1, frequency 36 lp/cm, and nominal selectivity of 11.9. It was mounted on the kV flat panel detector of an Elekta Synergy linear accelerator and tested in a phantom and a clinical study. Due to the flex of the linac and presence of gridline artifacts an angle dependent gain correction algorithm was devised to mitigate resulting artifacts. Scan reconstruction was performed using XVI4.5 augmented with inhouse developed image lag correction and Hounsfield unit calibration. To determine the necessary parameters for Hounsfield unit calibration and software scatter correction parameters, the Catphan 600 (The Phantom Laboratory) phantom was used. Image quality parameters were evaluated using CIRS CBCT Image Quality and Electron Density Phantom (CIRS) in two different geometries: one modeling head and neck and other pelvic region. Phantoms were acquired with and without the grid and reconstructed with and without software correction which was adapted for the different acquisition

Crystal Structure of the Dimeric Oct6 (Pou3fl) POU Domain Bound to Palindromic MORE DNA

Energy Technology Data Exchange (ETDEWEB)

R Jauch; S Choo; C Ng; P Kolatkar

2011-12-31

POU domains (named after their identification in Pit1, Oct1 unc86) are found in around 15 transcription factors encoded in mammalian genomes many of which feature prominently as key regulators at development bifurcations. For example, the POU III class Octamer binding protein 6 (Oct6) is expressed in embryonic stem cells and during neural development and drives the differentia5tion of myelinated cells in the central and peripheral nervous system. Defects in oct6 expression levels are linked to neurological disorders such as schizophrenia. POU proteins contain a bi-partite DNA binding domain that assembles on various DNA motifs with differentially configured subdomains. Intriguingly, alternative configurations of POU domains on different DNA sites were shown to affect the subsequent recruitment of transcriptional coactivators. Namely, binding of Oct1 to a Palindromic Oct-factor Recognition Element (PORE) was shown to facilitate the recruitment of the OBF1 coactivator whereas More of PORE (MORE) bound Oct1 does not. Moreover, Pit1 was shown to recruit the corepressor N-CoR only when bound to a variant MORE motif with a 2 bp half-site spacing. Therefore, POU proteins are seen as a paradigm for DNA induced allosteric effects on transcription factors modulating their regulatory potential. However, a big unresolved conundrum for the POU class and for most if not all other transcription factor classes is how highly similar proteins regulate different sets of genes causing fundamentally different biological responses. Ultimately, there must be subtle features enabling those factors to engage in contrasting molecular interactions in the cell. Thus, the dissection of the molecular details of the transcription-DNA recognition in general, and the formation of multimeric regulatory complexes, in particular, is highly desirable. To contribute to these efforts they solved the 2.05 {angstrom} crystal structure of Oct6 bound as a symmetrical homodimer to palindromic MORE DNA.

Species characterization in the genus Pestivirus according to palindromic nucleotide substitutions in the 5'-untranslated region.

Science.gov (United States)

Giangaspero, Massimo; Harasawa, Ryô

2011-06-01

The palindromic nucleotide substitutions (PNS) at the three variable loci (V1, V2 and V3) in the 5'-untranslated region (UTR) of the Pestivirus genome have been considered for taxonomical segregation of the species, through the evaluation of 534 strains. On the basis of qualitative and quantitative secondary structure characteristics, species have been identified within the genus, determining genetic distances between species isolates, clarifying borderline and multirelated sequences, and characterizing and clustering the Pestivirus strains showing unexpected genomic sequences. Nine genomic groups have been identified: the species Bovine viral diarrhea virus 1 (BVDV-1), Bovine viral diarrhea virus 2 (BVDV-2), Border disease virus (BDV) and Classical swine fever virus (CSFV) and the tentative species Pronghorn, Giraffe, Bovine viral diarrhea virus 3 (BVDV-3) (HoBi group), Border disease virus 2 (BDV-2) (Italian small ruminant isolates) and Bungowannah. Palindromic positions have been characterized according to changes in nucleotide base-pairs identifying low variable positions (LVP) including base-pairs present in less than 80% of the genus. The determination of divergence between single strain sequences or genetic groups was obtained easily by comparing base-pairing combinations from aligned secondary structures. This provided clear information such as the level of heterogeneity within a species, the relatedness between species, or facilitating the characterization and clustering of specific strains. The BVDV-1 and BDV species resulted heterogeneous, showing isolates located on a borderline in the species. Within the BVDV-2 species, two main genogroups were identified, with strains showing common sequence characteristics to both groups (multirelated strains). They could be allocated correctly by quantitative analysis. Similarly, the relation between CSFV and BDV species appeared very clearly. Also in this case, ambiguous strain sequences could be clustered in the

CRISPR/Cas9 Technology as an Emerging Tool for Targeting Amyotrophic Lateral Sclerosis (ALS).

Science.gov (United States)

Kruminis-Kaszkiel, Ewa; Juranek, Judyta; Maksymowicz, Wojciech; Wojtkiewicz, Joanna

2018-03-19

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) is a genome editing tool that has recently caught enormous attention due to its novelty, feasibility, and affordability. This system naturally functions as a defense mechanism in bacteria and has been repurposed as an RNA-guided DNA editing tool. Unlike zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 takes advantage of an RNA-guided DNA endonuclease enzyme, Cas9, which is able to generate double-strand breaks (DSBs) at specific genomic locations. It triggers cellular endogenous DNA repair pathways, contributing to the generation of desired modifications in the genome. The ability of the system to precisely disrupt DNA sequences has opened up new avenues in our understanding of amyotrophic lateral sclerosis (ALS) pathogenesis and the development of new therapeutic approaches. In this review, we discuss the current knowledge of the principles and limitations of the CRISPR/Cas9 system, as well as strategies to improve these limitations. Furthermore, we summarize novel approaches of engaging the CRISPR/Cas9 system in establishing an adequate model of neurodegenerative disease and in the treatment of SOD1-linked forms of ALS. We also highlight possible applications of this system in the therapy of ALS, both the inherited type as well as ALS of sporadic origin.

Emerging applications of genome-editing technology to examine functionality of GWAS-associated variants for complex traits.

Science.gov (United States)

Smith, Andrew J P; Deloukas, Panos; Munroe, Patricia B

2018-04-13

Over the last decade, genome-wide association studies (GWAS) have propelled the discovery of thousands of loci associated with complex diseases. The focus is now turning towards the function of these association signals, determining the causal variant(s) amongst those in strong linkage disequilibrium, and identifying their underlying mechanisms, such as long-range gene regulation. Genome-editing techniques utilising zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs) and clustered regularly-interspaced short palindromic repeats with Cas9 nuclease (CRISPR-Cas9), are becoming the tools of choice to establish functionality for these variants, due to the ability to assess effects of single variants in vivo. This review will discuss examples of how these technologies have begun to aid functional analysis of GWAS loci for complex traits such as cardiovascular disease, type 2 diabetes, cancer, obesity and autoimmune disease. We focus on analysis of variants occurring within non-coding genomic regions, as these comprise the majority of GWAS variants, providing the greatest challenges to determining functionality, and compare editing strategies that provide different levels of evidence for variant functionality. The review describes molecular insights into some of these potentially causal variants, and how these may relate to the pathology of the trait, and look towards future directions for these technologies in post-GWAS analysis, such as base-editing.

The commercialization of genome-editing technologies.

Science.gov (United States)

Brinegar, Katelyn; K Yetisen, Ali; Choi, Sun; Vallillo, Emily; Ruiz-Esparza, Guillermo U; Prabhakar, Anand M; Khademhosseini, Ali; Yun, Seok-Hyun

2017-11-01

The emergence of new gene-editing technologies is profoundly transforming human therapeutics, agriculture, and industrial biotechnology. Advances in clustered regularly interspaced short palindromic repeats (CRISPR) have created a fertile environment for mass-scale manufacturing of cost-effective products ranging from basic research to translational medicine. In our analyses, we evaluated the patent landscape of gene-editing technologies and found that in comparison to earlier gene-editing techniques, CRISPR has gained significant traction and this has established dominance. Although most of the gene-editing technologies originated from the industry, CRISPR has been pioneered by academic research institutions. The spinout of CRISPR biotechnology companies from academic institutions demonstrates a shift in entrepreneurship strategies that were previously led by the industry. These academic institutions, and their subsequent companies, are competing to generate comprehensive intellectual property portfolios to rapidly commercialize CRISPR products. Our analysis shows that the emergence of CRISPR has resulted in a fivefold increase in genome-editing bioenterprise investment over the last year. This entrepreneurial movement has spurred a global biotechnology revolution in the realization of novel gene-editing technologies. This global shift in bioenterprise will continue to grow as the demand for personalized medicine, genetically modified crops and environmentally sustainable biofuels increases. However, the monopolization of intellectual property, negative public perception of genetic engineering and ambiguous regulatory policies may limit the growth of these market segments.

Advances in genome editing for improved animal breeding: A review

Directory of Open Access Journals (Sweden)

Shakil Ahmad Bhat

2017-11-01

Full Text Available Since centuries, the traits for production and disease resistance are being targeted while improving the genetic merit of domestic animals, using conventional breeding programs such as inbreeding, outbreeding, or introduction of marker-assisted selection. The arrival of new scientific concepts, such as cloning and genome engineering, has added a new and promising research dimension to the existing animal breeding programs. Development of genome editing technologies such as transcription activator-like effector nuclease, zinc finger nuclease, and clustered regularly interspaced short palindromic repeats systems begun a fresh era of genome editing, through which any change in the genome, including specific DNA sequence or indels, can be made with unprecedented precision and specificity. Furthermore, it offers an opportunity of intensification in the frequency of desirable alleles in an animal population through gene-edited individuals more rapidly than conventional breeding. The specific research is evolving swiftly with a focus on improvement of economically important animal species or their traits all of which form an important subject of this review. It also discusses the hurdles to commercialization of these techniques despite several patent applications owing to the ambiguous legal status of genome-editing methods on account of their disputed classification. Nonetheless, barring ethical concerns gene-editing entailing economically important genes offers a tremendous potential for breeding animals with desirable traits.

Suppression or knockout of SaF/SaM overcomes the Sa-mediated hybrid male sterility in rice

Institute of Scientific and Technical Information of China (English)

Yongyao Xie; Baixiao Niu; Yunming Long; Gousi Li; Jintao Tang; Yaling Zhang; Ding Ren; Yao-Guang Liu; Letian Chen

2017-01-01

Hybrids between the indica and japonica subspecies of rice (Oryza sativa) are usually sterile, which hinders utilization of heterosis in the inter-subspecific hybrid breeding. The complex locus Sa comprises two adjacently located genes, SaF and SaM, which interact to cause abortion of pollen grains carrying the japonica allele in japonica-indica hybrids. Here we showed that silencing of SaF or SaM by RNA interference restored male fertility in indica-japonica hybrids with heterozygous Sa. We further used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing to knockout the SaF and SaM alleles, respectively, of an indica rice line to create hybrid-compatible lines. The resultant artificial neutral alleles did not affect pollen viability and other agricultural traits, but did break down the reproductive barrier in the hybrids. We found that some rice lines have natural neutral allele Sa-n, which was compatible with the typical japonica or indica Sa alleles in hybrids. Our results demonstrate that SaF and SaM are required for hybrid male sterility, but are not essential for pollen development. This study provides effective approaches for the generation of hybrid-compatible lines by knocking out the Sa locus or using the natural Sa-n allele to overcome hybrid male sterility in rice breeding.

Heterologous and endogenous U6 snRNA promoters enable CRISPR/Cas9 mediated genome editing in Aspergillus niger.

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Zheng, Xiaomei; Zheng, Ping; Sun, Jibin; Kun, Zhang; Ma, Yanhe

2018-01-01

U6 promoters have been used for single guide RNA (sgRNA) transcription in the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas9) genome editing system. However, no available U6 promoters have been identified in Aspergillus niger, which is an important industrial platform for organic acid and protein production. Two CRISPR/Cas9 systems established in A. niger have recourse to the RNA polymerase II promoter or in vitro transcription for sgRNA synthesis, but these approaches generally increase cloning efforts and genetic manipulation. The validation of functional RNA polymerase II promoters is therefore an urgent need for A. niger . Here, we developed a novel CRISPR/Cas9 system in A. niger for sgRNA expression, based on one endogenous U6 promoter and two heterologous U6 promoters. The three tested U6 promoters enabled sgRNA transcription and the disruption of the polyketide synthase albA gene in A. niger . Furthermore, this system enabled highly efficient gene insertion at the targeted genome loci in A. niger using donor DNAs with homologous arms as short as 40-bp. This study demonstrated that both heterologous and endogenous U6 promoters were functional for sgRNA expression in A. niger . Based on this result, a novel and simple CRISPR/Cas9 toolbox was established in A. niger, that will benefit future gene functional analysis and genome editing.

Engineering Plant Immunity: Using CRISPR/Cas9 to Generate Virus Resistance

KAUST Repository

Zaidi, Syed Shan-e-Ali

2016-11-08

Plant viruses infect many economically important crops, including wheat, cotton, maize, cassava, and other vegetables. These viruses pose a serious threat to agriculture worldwide, as decreases in cropland area per capita may cause production to fall short of that required to feed the increasing world population. Under these circumstances, conventional strategies can fail to control rapidly evolving and emerging plant viruses. Genome-engineering strategies have recently emerged as promising tools to introduce desirable traits in many eukaryotic species, including plants. Among these genome engineering technologies, the CRISPR (clustered regularly interspaced palindromic repeats)/CRISPR-associated 9 (CRISPR/Cas9) system has received special interest because of its simplicity, efficiency, and reproducibility. Recent studies have used CRISPR/Cas9 to engineer virus resistance in plants, either by directly targeting and cleaving the viral genome, or by modifying the host plant genome to introduce viral immunity. Here, we briefly describe the biology of the CRISPR/Cas9 system and plant viruses, and how different genome engineering technologies have been used to target these viruses. We further describe the main findings from recent studies of CRISPR/Cas9-mediated viral interference and discuss how these findings can be applied to improve global agriculture. We conclude by pinpointing the gaps in our knowledge and the outstanding questions regarding CRISPR/Cas9-mediated viral immunity.

CRISPR-mediated genotypic and phenotypic correction of a chronic granulomatous disease mutation in human iPS cells

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Flynn, Rowan; Grundmann, Alexander; Renz, Peter; Hänseler, Walther; James, William S.; Cowley, Sally A.; Moore, Michael D.

2015-01-01

Chronic granulomatous disease (CGD) is a rare genetic disease characterized by severe and persistent childhood infections. It is caused by the lack of an antipathogen oxidative burst, normally performed by phagocytic cells to contain and clear bacterial and fungal growth. Restoration of immune function can be achieved with heterologous bone marrow transplantation; however, autologous bone marrow transplantation would be a preferable option. Thus, a method is required to recapitulate the function of the diseased gene within the patient's own cells. Gene therapy approaches for CGD have employed randomly integrating viruses with concomitant issues of insertional mutagenesis, inaccurate gene dosage, and gene silencing. Here, we explore the potential of the recently described clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 site-specific nuclease system to encourage repair of the endogenous gene by enhancing the levels of homologous recombination. Using induced pluripotent stem cells derived from a CGD patient containing a single intronic mutation in the CYBB gene, we show that footprintless gene editing is a viable option to correct disease mutations. Gene correction results in restoration of oxidative burst function in iPS-derived phagocytes by reintroduction of a previously skipped exon in the cytochrome b-245 heavy chain (CYBB) protein. This study provides proof-of-principle for a gene therapy approach to CGD treatment using CRISPR-Cas9. PMID:26101162

CRISPR Correction of a Homozygous Low-Density Lipoprotein Receptor Mutation in Familial Hypercholesterolemia Induced Pluripotent Stem Cells.

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Omer, Linda; Hudson, Elizabeth A; Zheng, Shirong; Hoying, James B; Shan, Yuan; Boyd, Nolan L

2017-11-01

Familial hypercholesterolemia (FH) is a hereditary disease primarily due to mutations in the low-density lipoprotein receptor (LDLR) that lead to elevated cholesterol and premature development of cardiovascular disease. Homozygous FH patients (HoFH) with two dysfunctional LDLR alleles are not as successfully treated with standard hypercholesterol therapies, and more aggressive therapeutic approaches to control cholesterol levels must be considered. Liver transplant can resolve HoFH, and hepatocyte transplantation has shown promising results in animals and humans. However, demand for donated livers and high-quality hepatocytes overwhelm the supply. Human pluripotent stem cells can differentiate to hepatocyte-like cells (HLCs) with the potential for experimental and clinical use. To be of future clinical use as autologous cells, LDLR genetic mutations in derived FH-HLCs need to be corrected. Genome editing technology clustered-regularly-interspaced-short-palindromic-repeats/CRISPR-associated 9 (CRISPR/Cas9) can repair pathologic genetic mutations in human induced pluripotent stem cells. We used CRISPR/Cas9 genome editing to permanently correct a 3-base pair homozygous deletion in LDLR exon 4 of patient-derived HoFH induced pluripotent stem cells. The genetic correction restored LDLR-mediated endocytosis in FH-HLCs and demonstrates the proof-of-principle that CRISPR-mediated genetic modification can be successfully used to normalize HoFH cholesterol metabolism deficiency at the cellular level.

A Prospective Treatment Option for Lysosomal Storage Diseases: CRISPR/Cas9 Gene Editing Technology for Mutation Correction in Induced Pluripotent Stem Cells.

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Christensen, Chloe L; Choy, Francis Y M

2017-02-24

Ease of design, relatively low cost and a multitude of gene-altering capabilities have all led to the adoption of the sophisticated and yet simple gene editing system: clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9). The CRISPR/Cas9 system holds promise for the correction of deleterious mutations by taking advantage of the homology directed repair pathway and by supplying a correction template to the affected patient's cells. Currently, this technique is being applied in vitro in human-induced pluripotent stem cells (iPSCs) to correct a variety of severe genetic diseases, but has not as of yet been used in iPSCs derived from patients affected with a lysosomal storage disease (LSD). If adopted into clinical practice, corrected iPSCs derived from cells that originate from the patient themselves could be used for therapeutic amelioration of LSD symptoms without the risks associated with allogeneic stem cell transplantation. CRISPR/Cas9 editing in a patient's cells would overcome the costly, lifelong process associated with currently available treatment methods, including enzyme replacement and substrate reduction therapies. In this review, the overall utility of the CRISPR/Cas9 gene editing technique for treatment of genetic diseases, the potential for the treatment of LSDs and methods currently employed to increase the efficiency of this re-engineered biological system will be discussed.

Improved hematopoietic differentiation efficiency of gene-corrected beta-thalassemia induced pluripotent stem cells by CRISPR/Cas9 system.

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Song, Bing; Fan, Yong; He, Wenyin; Zhu, Detu; Niu, Xiaohua; Wang, Ding; Ou, Zhanhui; Luo, Min; Sun, Xiaofang

2015-05-01

The generation of beta-thalassemia (β-Thal) patient-specific induced pluripotent stem cells (iPSCs), subsequent homologous recombination-based gene correction of disease-causing mutations/deletions in the β-globin gene (HBB), and their derived hematopoietic stem cell (HSC) transplantation offers an ideal therapeutic solution for treating this disease. However, the hematopoietic differentiation efficiency of gene-corrected β-Thal iPSCs has not been well evaluated in the previous studies. In this study, we used the latest gene-editing tool, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), to correct β-Thal iPSCs; gene-corrected cells exhibit normal karyotypes and full pluripotency as human embryonic stem cells (hESCs) showed no off-targeting effects. Then, we evaluated the differentiation efficiency of the gene-corrected β-Thal iPSCs. We found that during hematopoietic differentiation, gene-corrected β-Thal iPSCs showed an increased embryoid body ratio and various hematopoietic progenitor cell percentages. More importantly, the gene-corrected β-Thal iPSC lines restored HBB expression and reduced reactive oxygen species production compared with the uncorrected group. Our study suggested that hematopoietic differentiation efficiency of β-Thal iPSCs was greatly improved once corrected by the CRISPR/Cas9 system, and the information gained from our study would greatly promote the clinical application of β-Thal iPSC-derived HSCs in transplantation.

Temperature-dependent sex-reversal by a transformer-2 gene-edited mutation in the spotted wing drosophila, Drosophila suzukii.

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Li, Jianwei; Handler, Alfred M

2017-09-28

Female to male sex reversal was achieved in an emerging agricultural insect pest, Drosophila suzukii, by creating a temperature-sensitive point mutation in the sex-determination gene, transformer-2 (tra-2), using CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) homology-directed repair gene-editing. Ds-tra-2 ts2 mutants developed as normal fertile XX and XY adults at permissive temperatures below 20 °C, but at higher restrictive temperatures (26 to 29 °C) chromosomal XX females developed as sterile intersexuals with a predominant male phenotype, while XY males developed with normal morphology, but were sterile. The temperature-dependent function of the Ds-TRA-2 ts2 protein was also evident by the up- and down-regulation of female-specific Ds-Yolk protein 1 (Ds-Yp1) gene expression by temperature shifts during adulthood. This study confirmed the temperature-dependent function of a gene-edited mutation and provides a new method for the more general creation of conditional mutations for functional genomic analysis in insects, and other organisms. Furthermore, it provides a temperature-dependent system for creating sterile male populations useful for enhancing the efficacy of biologically-based programs, such as the sterile insect technique (SIT), to control D. suzukii and other insect pest species of agricultural and medical importance.

Identification of Human Junctional Adhesion Molecule 1 as a Functional Receptor for the Hom-1 Calicivirus on Human Cells

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Stanislav V. Sosnovtsev

2017-02-01

Full Text Available The Hom-1 vesivirus was reported in 1998 following the inadvertent transmission of the animal calicivirus San Miguel sea lion virus to a human host in a laboratory. We characterized the Hom-1 strain and investigated the mechanism by which human cells could be infected. An expression library of 3,559 human plasma membrane proteins was screened for reactivity with Hom-1 virus-like particles, and a single interacting protein, human junctional adhesion molecule 1 (hJAM1, was identified. Transient expression of hJAM1 conferred susceptibility to Hom-1 infection on nonpermissive Chinese hamster ovary (CHO cells. Virus infection was markedly inhibited when CHO cells stably expressing hJAM were pretreated with anti-hJAM1 monoclonal antibodies. Cell lines of human origin were tested for growth of Hom-1, and efficient replication was observed in HepG2, HuH7, and SK-CO15 cells. The three cell lines (of hepatic or intestinal origin were confirmed to express hJAM1 on their surface, and clustered regularly interspaced short palindromic repeats/Cas9-mediated knockout of the hJAM1 gene in each line abolished Hom-1 propagation. Taken together, our data indicate that entry of the Hom-1 vesivirus into these permissive human cell lines is mediated by the plasma membrane protein hJAM1 as a functional receptor.

Non-viral delivery systems for CRISPR/Cas9-based genome editing: Challenges and opportunities.

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Li, Ling; Hu, Shuo; Chen, Xiaoyuan

2018-07-01

In recent years, CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) genome editing systems have become one of the most robust platforms in basic biomedical research and therapeutic applications. To date, efficient in vivo delivery of the CRISPR/Cas9 system to the targeted cells remains a challenge. Although viral vectors have been widely used in the delivery of the CRISPR/Cas9 system in vitro and in vivo, their fundamental shortcomings, such as the risk of carcinogenesis, limited insertion size, immune responses and difficulty in large-scale production, severely limit their further applications. Alternative non-viral delivery systems for CRISPR/Cas9 are urgently needed. With the rapid development of non-viral vectors, lipid- or polymer-based nanocarriers have shown great potential for CRISPR/Cas9 delivery. In this review, we analyze the pros and cons of delivering CRISPR/Cas9 systems in the form of plasmid, mRNA, or protein and then discuss the limitations and challenges of CRISPR/Cas9-based genome editing. Furthermore, current non-viral vectors that have been applied for CRISPR/Cas9 delivery in vitro and in vivo are outlined in details. Finally, critical obstacles for non-viral delivery of CRISPR/Cas9 system are highlighted and promising strategies to overcome these barriers are proposed. Published by Elsevier Ltd.

CRISPR/Cas9: the Jedi against the dark empire of diseases.

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Khan, Sehrish; Mahmood, Muhammad Shahid; Rahman, Sajjad Ur; Zafar, Hassan; Habibullah, Sultan; Khan, Zulqarnain; Ahmad, Aftab

2018-03-28

Advances in Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated system (CRISPR/Cas9) has dramatically reshaped our ability to edit genomes. The scientific community is using CRISPR/Cas9 for various biotechnological and medical purposes. One of its most important uses is developing potential therapeutic strategies against diseases. CRISPR/Cas9 based approaches have been increasingly applied to the treatment of human diseases like cancer, genetic, immunological and neurological disorders and viral diseases. These strategies using CRISPR/Cas9 are not only therapy oriented but can also be used for disease modeling as well, which in turn can lead to the improved understanding of mechanisms of various infectious and genetic diseases. In addition, CRISPR/Cas9 system can also be used as programmable antibiotics to kill the bacteria sequence specifically and therefore can bypass multidrug resistance. Furthermore, CRISPR/Cas9 based gene drive may also hold the potential to limit the spread of vector borne diseases. This bacterial and archaeal adaptive immune system might be a therapeutic answer to previous incurable diseases, of course rigorous testing is required to corroborate these claims. In this review, we provide an insight about the recent developments using CRISPR/Cas9 against various diseases with respect to disease modeling and treatment, and what future perspectives should be noted while using this technology.

Efficient genome editing by FACS enrichment of paired D10A Cas9 nickases coupled with fluorescent proteins.

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Gopalappa, Ramu; Song, Myungjae; Chandrasekaran, Arun Pandian; Das, Soumyadip; Haq, Saba; Koh, Hyun Chul; Ramakrishna, Suresh

2018-05-31

Targeted genome editing by clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) raised concerns over off-target effects. The use of double-nicking strategy using paired Cas9 nickase has been developed to minimize off-target effects. However, it was reported that the efficiency of paired nickases were comparable or lower than that of either corresponding nuclease alone. Recently, we conducted a systematic comparison of the efficiencies of several paired Cas9 with their corresponding Cas9 nucleases and showed that paired D10A Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption. However, sometimes the designed paired Cas9 nickases exhibited significantly lower mutation frequencies than nucleases, hampering the generation of cells containing paired Cas9 nickase-induced mutations. Here we implemented IRES peptide-conjugation of fluorescent protein to Cas9 nickase and subjected for fluorescence-activated cell sorting. The sorted cell populations are highly enriched with cells containing paired Cas9 nickase-induced mutations, by a factor of up to 40-fold as compared with the unsorted population. Furthermore, gene-disrupted single cell clones using paired nickases followed by FACS sorting strategy were generated highly efficiently, without compromising with its low off-target effects. We envision that our fluorescent protein coupled paired nickase-mediated gene disruption, facilitating efficient and highly specific genome editing in medical research.

Ecological and genetic interactions between cyanobacteria and viruses in a low-oxygen mat community inferred through metagenomics and metatranscriptomics.

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Voorhies, Alexander A; Eisenlord, Sarah D; Marcus, Daniel N; Duhaime, Melissa B; Biddanda, Bopaiah A; Cavalcoli, James D; Dick, Gregory J

2016-02-01

Metagenomic and metatranscriptomic sequencing was conducted on cyanobacterial mats of the Middle Island Sinkhole (MIS), Lake Huron. Metagenomic data from 14 samples collected over 5 years were used to reconstruct genomes of two genotypes of a novel virus, designated PhV1 type A and PhV1 type B. Both viral genotypes encode and express nblA, a gene involved in degrading phycobilisomes, which are complexes of pigmented proteins that harvest light for photosynthesis. Phylogenetic analysis indicated that the viral-encoded nblA is derived from the host cyanobacterium, Phormidium MIS-PhA. The cyanobacterial host also has two complete CRISPR (clustered regularly interspaced short palindromic repeats) systems that serve as defence mechanisms for bacteria and archaea against viruses and plasmids. One 45 bp CRISPR spacer from Phormidium had 100% nucleotide identity to PhV1 type B, but this region was absent from PhV1 type A. Transcripts from PhV1 and the Phormidium CRISPR loci were detected in all six metatranscriptomic data sets (three during the day and three at night), indicating that both are transcriptionally active in the environment. These results reveal ecological and genetic interactions between viruses and cyanobacteria at MIS, highlighting the value of parallel analysis of viruses and hosts in understanding ecological interactions in natural communities. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Application of CRISPR/Cas9 Genome Editing Technology for the Improvement of Crops Cultivated in Tropical Climates: Recent Progress, Prospects, and Challenges

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Effi Haque

2018-05-01

Full Text Available The world population is expected to increase from 7.3 to 9.7 billion by 2050. Pest outbreak and increased abiotic stresses due to climate change pose a high risk to tropical crop production. Although conventional breeding techniques have significantly increased crop production and yield, new approaches are required to further improve crop production in order to meet the global growing demand for food. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9 (CRISPR-associated protein9 genome editing technology has shown great promise for quickly addressing emerging challenges in agriculture. It can be used to precisely modify genome sequence of any organism including plants to achieve the desired trait. Compared to other genome editing tools such as zinc finger nucleases (ZFNs and transcriptional activator-like effector nucleases (TALENs, CRISPR/Cas9 is faster, cheaper, precise and highly efficient in editing genomes even at the multiplex level. Application of CRISPR/Cas9 technology in editing the plant genome is emerging rapidly. The CRISPR/Cas9 is becoming a user-friendly tool for development of non-transgenic genome edited crop plants to counteract harmful effects from climate change and ensure future food security of increasing population in tropical countries. This review updates current knowledge and potentials of CRISPR/Cas9 for improvement of crops cultivated in tropical climates to gain resiliency against emerging pests and abiotic stresses.

The Adaptive Immune System of Haloferax volcanii

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Lisa-Katharina Maier

2015-02-01

Full Text Available To fight off invading genetic elements, prokaryotes have developed an elaborate defence system that is both adaptable and heritable—the CRISPR-Cas system (CRISPR is short for: clustered regularly interspaced short palindromic repeats and Cas: CRISPR associated. Comprised of proteins and multiple small RNAs, this prokaryotic defence system is present in 90% of archaeal and 40% of bacterial species, and enables foreign intruders to be eliminated in a sequence-specific manner. There are three major types (I–III and at least 14 subtypes of this system, with only some of the subtypes having been analysed in detail, and many aspects of the defence reaction remaining to be elucidated. Few archaeal examples have so far been analysed. Here we summarize the characteristics of the CRISPR-Cas system of Haloferax volcanii, an extremely halophilic archaeon originally isolated from the Dead Sea. It carries a single CRISPR-Cas system of type I-B, with a Cascade like complex composed of Cas proteins Cas5, Cas6b and Cas7. Cas6b is essential for CRISPR RNA (crRNA maturation but is otherwise not required for the defence reaction. A systematic search revealed that six protospacer adjacent motif (PAM sequences are recognised by the Haloferax defence system. For successful invader recognition, a non-contiguous seed sequence of 10 base-pairs between the crRNA and the invader is required.

The CRISPR/Cas genome-editing tool: application in improvement of crops

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SURENDER eKHATODIA

2016-04-01

Full Text Available The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR associated Cas9/sgRNA system is a novel fledgling targeted genome-editing technique from bacterial immune system, which is a cheap, easy and most rapidly adopted genome editing tool transforming to revolutionary paradigm. Cas9 protein is an RNA guided endonuclease utilized for creating targeted double stranded breaks with only a short RNA sequence to confer recognition of the target in animals and plants. Development of genetically edited (GE crops similar to those developed by conventional or mutation breeding using this potential technique makes it a promising and extremely versatile tool for providing sustainable productive agriculture for better feeding of rapidly growing population in changing climate. The emerging areas of research for the genome editing in plants are like, interrogating gene function, rewiring the regulatory signaling networks, sgRNA library for high-throughput loss-of-function screening. In this review, we will discuss the broad applicability of the Cas9 nuclease mediated targeted plant genome editing for development of designer crops. The regulatory uncertainty and social acceptance of plant breeding by Cas9 genome editing have also been discussed. The non-GM designer genetically edited plants could prospect climate resilient and sustainable energy agriculture in coming future for maximizing the yield by combating abiotic and biotic stresses with this new innovative plant breeding technique.

Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR proteins.

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Bondy-Denomy, Joseph; Garcia, Bianca; Strum, Scott; Du, Mingjian; Rollins, MaryClare F; Hidalgo-Reyes, Yurima; Wiedenheft, Blake; Maxwell, Karen L; Davidson, Alan R

2015-10-01

The battle for survival between bacteria and the viruses that infect them (phages) has led to the evolution of many bacterial defence systems and phage-encoded antagonists of these systems. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated (cas) genes comprise an adaptive immune system that is one of the most widespread means by which bacteria defend themselves against phages. We identified the first examples of proteins produced by phages that inhibit a CRISPR-Cas system. Here we performed biochemical and in vivo investigations of three of these anti-CRISPR proteins, and show that each inhibits CRISPR-Cas activity through a distinct mechanism. Two block the DNA-binding activity of the CRISPR-Cas complex, yet do this by interacting with different protein subunits, and using steric or non-steric modes of inhibition. The third anti-CRISPR protein operates by binding to the Cas3 helicase-nuclease and preventing its recruitment to the DNA-bound CRISPR-Cas complex. In vivo, this anti-CRISPR can convert the CRISPR-Cas system into a transcriptional repressor, providing the first example-to our knowledge-of modulation of CRISPR-Cas activity by a protein interactor. The diverse sequences and mechanisms of action of these anti-CRISPR proteins imply an independent evolution, and foreshadow the existence of other means by which proteins may alter CRISPR-Cas function.

In silico genomic insights into aspects of food safety and defense mechanisms of a potentially probiotic Lactobacillus pentosus MP-10 isolated from brines of naturally fermented Aloreña green table olives.

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Hikmate Abriouel

Full Text Available Lactobacillus pentosus MP-10, isolated from brines of naturally fermented Aloreña green table olives, exhibited high probiotic potential. The genome sequence of L. pentosus MP-10 is currently considered the largest genome among lactobacilli, highlighting the microorganism's ecological flexibility and adaptability. Here, we analyzed the complete genome sequence for the presence of acquired antibiotic resistance and virulence determinants to understand their defense mechanisms and explore its putative safety in food. The annotated genome sequence revealed evidence of diverse mobile genetic elements, such as prophages, transposases and transposons involved in their adaptation to brine-associated niches. In-silico analysis of L. pentosus MP-10 genome sequence identified a CRISPR (clustered regularly interspaced short palindromic repeats/cas (CRISPR-associated protein genes as an immune system against foreign genetic elements, which consisted of six arrays (4-12 repeats and eleven predicted cas genes [CRISPR1 and CRISPR2 consisted of 3 (Type II-C and 8 (Type I genes] with high similarity to L. pentosus KCA1. Bioinformatic analyses revealed L. pentosus MP-10 to be absent of acquired antibiotic resistance genes, and most resistance genes were related to efflux mechanisms; no virulence determinants were found in the genome. This suggests that L. pentosus MP-10 could be considered safe and with high-adaptation potential, which could facilitate its application as a starter culture and probiotic in food preparations.

Crystal structure of the Csm3-Csm4 subcomplex in the type III-A CRISPR-Cas interference complex.

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Numata, Tomoyuki; Inanaga, Hideko; Sato, Chikara; Osawa, Takuo

2015-01-30

Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci play a pivotal role in the prokaryotic host defense system against invading genetic materials. The CRISPR loci are transcribed to produce CRISPR RNAs (crRNAs), which form interference complexes with CRISPR-associated (Cas) proteins to target the invading nucleic acid for degradation. The interference complex of the type III-A CRISPR-Cas system is composed of five Cas proteins (Csm1-Csm5) and a crRNA, and targets invading DNA. Here, we show that the Csm1, Csm3, and Csm4 proteins from Methanocaldococcus jannaschii form a stable subcomplex. We also report the crystal structure of the M. jannaschii Csm3-Csm4 subcomplex at 3.1Å resolution. The complex structure revealed the presence of a basic concave surface around their interface, suggesting the RNA and/or DNA binding ability of the complex. A gel retardation analysis showed that the Csm3-Csm4 complex binds single-stranded RNA in a non-sequence-specific manner. Csm4 structurally resembles Cmr3, a component of the type III-B CRISPR-Cas interference complex. Based on bioinformatics, we constructed a model structure of the Csm1-Csm4-Csm3 ternary complex, which provides insights into its role in the Csm interference complex. Copyright © 2014 Elsevier Ltd. All rights reserved.

Interference activity of a minimal Type I CRISPR–Cas system from Shewanella putrefaciens

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Dwarakanath, Srivatsa; Brenzinger, Susanne; Gleditzsch, Daniel; Plagens, André; Klingl, Andreas; Thormann, Kai; Randau, Lennart

2015-01-01

Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas (CRISPR-associated) systems exist in bacterial and archaeal organisms and provide immunity against foreign DNA. The Cas protein content of the DNA interference complexes (termed Cascade) varies between different CRISPR-Cas subtypes. A minimal variant of the Type I-F system was identified in proteobacterial species including Shewanella putrefaciens CN-32. This variant lacks a large subunit (Csy1), Csy2 and Csy3 and contains two unclassified cas genes. The genome of S. putrefaciens CN-32 contains only five Cas proteins (Cas1, Cas3, Cas6f, Cas1821 and Cas1822) and a single CRISPR array with 81 spacers. RNA-Seq analyses revealed the transcription of this array and the maturation of crRNAs (CRISPR RNAs). Interference assays based on plasmid conjugation demonstrated that this CRISPR-Cas system is active in vivo and that activity is dependent on the recognition of the dinucleotide GG PAM (Protospacer Adjacent Motif) sequence and crRNA abundance. The deletion of cas1821 and cas1822 reduced the cellular crRNA pool. Recombinant Cas1821 was shown to form helical filaments bound to RNA molecules, which suggests its role as the Cascade backbone protein. A Cascade complex was isolated which contained multiple Cas1821 copies, Cas1822, Cas6f and mature crRNAs. PMID:26350210

A type III-B CRISPR-Cas effector complex mediating massive target DNA destruction.

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Han, Wenyuan; Li, Yingjun; Deng, Ling; Feng, Mingxia; Peng, Wenfang; Hallstrøm, Søren; Zhang, Jing; Peng, Nan; Liang, Yun Xiang; White, Malcolm F; She, Qunxin

2017-02-28

The CRISPR (clustered regularly interspaced short palindromic repeats) system protects archaea and bacteria by eliminating nucleic acid invaders in a crRNA-guided manner. The Sulfolobus islandicus type III-B Cmr-α system targets invading nucleic acid at both RNA and DNA levels and DNA targeting relies on the directional transcription of the protospacer in vivo. To gain further insight into the involved mechanism, we purified a native effector complex of III-B Cmr-α from S. islandicus and characterized it in vitro. Cmr-α cleaved RNAs complementary to crRNA present in the complex and its ssDNA destruction activity was activated by target RNA. The ssDNA cleavage required mismatches between the 5΄-tag of crRNA and the 3΄-flanking region of target RNA. An invader plasmid assay showed that mutation either in the histidine-aspartate acid (HD) domain (a quadruple mutation) or in the GGDD motif of the Cmr-2α protein resulted in attenuation of the DNA interference in vivo. However, double mutation of the HD motif only abolished the DNase activity in vitro. Furthermore, the activated Cmr-α binary complex functioned as a highly active DNase to destroy a large excess DNA substrate, which could provide a powerful means to rapidly degrade replicating viral DNA. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

COSMID: A Web-based Tool for Identifying and Validating CRISPR/Cas Off-target Sites

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Thomas J Cradick

2014-01-01

Full Text Available Precise genome editing using engineered nucleases can significantly facilitate biological studies and disease treatment. In particular, clustered regularly interspaced short palindromic repeats (CRISPR with CRISPR-associated (Cas proteins are a potentially powerful tool for modifying a genome by targeted cleavage of DNA sequences complementary to designed guide strand RNAs. Although CRISPR/Cas systems can have on-target cleavage rates close to the transfection rates, they may also have relatively high off-target cleavage at similar genomic sites that contain one or more base pair mismatches, and insertions or deletions relative to the guide strand. We have developed a bioinformatics-based tool, COSMID (CRISPR Off-target Sites with Mismatches, Insertions, and Deletions that searches genomes for potential off-target sites (http://crispr.bme.gatech.edu. Based on the user-supplied guide strand and input parameters, COSMID identifies potential off-target sites with the specified number of mismatched bases and insertions or deletions when compared with the guide strand. For each site, amplification primers optimal for the chosen application are also given as output. This ranked-list of potential off-target sites assists the choice and evaluation of intended target sites, thus helping the design of CRISPR/Cas systems with minimal off-target effects, as well as the identification and quantification of CRISPR/Cas induced off-target cleavage in cells.

Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis

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Keisuke Nakajima

2015-01-01

Full Text Available Zinc-finger nucleases, transcription activator-like effector nucleases (TALENs and the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins system are potentially powerful tools for producing tailor-made knockout animals. However, their mutagenic activity is not high enough to induce mutations at all loci of a target gene throughout an entire tadpole. In this study, we present a highly efficient method for introducing gene modifications at almost all target sequences in randomly selected embryos. The gene modification activity of TALEN is enhanced by adopting the host-transfer technique. In our method, the efficiency is further improved by injecting TALEN mRNAs fused to the 3′UTR of the Xenopus DEADSouth gene into oocytes, which are then transferred into a host female frog, where they are ovulated and fertilized. The addition of the 3′UTR of the DEADSouth gene promotes mRNA translation in the oocytes and increases the expression of TALEN proteins to near-maximal levels three hours post fertilization (hpf. In contrast, TALEN mRNAs without this 3′UTR are translated infrequently in oocytes. Our data suggest that genomic DNA is more sensitive to TALEN proteins from fertilization to the midblastula (MBT stage. Our method works by increasing the levels of TALEN proteins during the pre-MBT stages.

Bioinformatic prediction and functional characterization of human KIAA0100 gene

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He Cui

2017-02-01

Full Text Available Our previous study demonstrated that human KIAA0100 gene was a novel acute monocytic leukemia-associated antigen (MLAA gene. But the functional characterization of human KIAA0100 gene has remained unknown to date. Here, firstly, bioinformatic prediction of human KIAA0100 gene was carried out using online softwares; Secondly, Human KIAA0100 gene expression was downregulated by the clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR-associated (Cas 9 system in U937 cells. Cell proliferation and apoptosis were next evaluated in KIAA0100-knockdown U937 cells. The bioinformatic prediction showed that human KIAA0100 gene was located on 17q11.2, and human KIAA0100 protein was located in the secretory pathway. Besides, human KIAA0100 protein contained a signalpeptide, a transmembrane region, three types of secondary structures (alpha helix, extended strand, and random coil , and four domains from mitochondrial protein 27 (FMP27. The observation on functional characterization of human KIAA0100 gene revealed that its downregulation inhibited cell proliferation, and promoted cell apoptosis in U937 cells. To summarize, these results suggest human KIAA0100 gene possibly comes within mitochondrial genome; moreover, it is a novel anti-apoptotic factor related to carcinogenesis or progression in acute monocytic leukemia, and may be a potential target for immunotherapy against acute monocytic leukemia.

A Prospective Treatment Option for Lysosomal Storage Diseases: CRISPR/Cas9 Gene Editing Technology for Mutation Correction in Induced Pluripotent Stem Cells

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Chloe L. Christensen

2017-02-01

Full Text Available Ease of design, relatively low cost and a multitude of gene-altering capabilities have all led to the adoption of the sophisticated and yet simple gene editing system: clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9. The CRISPR/Cas9 system holds promise for the correction of deleterious mutations by taking advantage of the homology directed repair pathway and by supplying a correction template to the affected patient’s cells. Currently, this technique is being applied in vitro in human-induced pluripotent stem cells (iPSCs to correct a variety of severe genetic diseases, but has not as of yet been used in iPSCs derived from patients affected with a lysosomal storage disease (LSD. If adopted into clinical practice, corrected iPSCs derived from cells that originate from the patient themselves could be used for therapeutic amelioration of LSD symptoms without the risks associated with allogeneic stem cell transplantation. CRISPR/Cas9 editing in a patient’s cells would overcome the costly, lifelong process associated with currently available treatment methods, including enzyme replacement and substrate reduction therapies. In this review, the overall utility of the CRISPR/Cas9 gene editing technique for treatment of genetic diseases, the potential for the treatment of LSDs and methods currently employed to increase the efficiency of this re-engineered biological system will be discussed.

The highly dynamic CRISPR1 system of Streptococcus agalactiae controls the diversity of its mobilome.

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Lopez-Sanchez, Maria-José; Sauvage, Elisabeth; Da Cunha, Violette; Clermont, Dominique; Ratsima Hariniaina, Elisoa; Gonzalez-Zorn, Bruno; Poyart, Claire; Rosinski-Chupin, Isabelle; Glaser, Philippe

2012-09-01

Clustered regularly interspaced short palindromic repeats (CRISPR) confer immunity against mobile genetic elements (MGEs) in prokaryotes. Streptococcus agalactiae, a leading cause of neonatal infections contains in its genome two CRISPR/Cas systems. We show that type 1-C CRISPR2 is present in few strains but type 2-A CRISPR1 is ubiquitous. Comparative sequence analysis of the CRISPR1 spacer content of 351 S. agalactiae strains revealed that it is extremely diverse due to the acquisition of new spacers, spacer duplications and spacer deletions that witness the dynamics of this system. The spacer content profile mirrors the S. agalactiae population structure. Transfer of a conjugative transposon targeted by CRISPR1 selected for spacer rearrangements, suggesting that deletions and duplications pre-exist in the population. The comparison of protospacers located within MGE or the core genome and protospacer-associated motif-shuffling demonstrated that the GG motif is sufficient to discriminate self and non-self and for spacer selection and integration. Strikingly more than 40% of the 949 different CRISPR1 spacers identified target MGEs found in S. agalactiae genomes. We thus propose that the S. agalactiae type II-A CRISPR1/Cas system modulates the cohabitation of the species with its mobilome, as such contributing to the diversity of MGEs in the population. © 2012 Blackwell Publishing Ltd.

Mapping of Wnt-Frizzled interactions by multiplex CRISPR targeting of receptor gene families.

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Voloshanenko, Oksana; Gmach, Philipp; Winter, Jan; Kranz, Dominique; Boutros, Michael

2017-11-01

Signaling pathway modules are often encoded by several closely related paralogous genes that can have redundant roles and are therefore difficult to analyze by loss-of-function analysis. A typical example is the Wnt signaling pathway, which in mammals is mediated by 19 Wnt ligands that can bind to 10 Frizzled (FZD) receptors. Although significant progress in understanding Wnt-FZD receptor interactions has been made in recent years, tools to generate systematic interaction maps have been largely lacking. Here we generated cell lines with multiplex mutant alleles of FZD1 , FZD2 , and FZD7 and demonstrate that these cells are unresponsive to canonical Wnt ligands. Subsequently, we performed genetic rescue experiments with combinations of FZDs and canonical Wnts to create a functional ligand-receptor interaction map. These experiments showed that whereas several Wnt ligands, such as Wnt3a, induce signaling through a broad spectrum of FZD receptors, others, such as Wnt8a, act through a restricted set of FZD genes. Together, our results map functional interactions of FZDs and 10 Wnt ligands and demonstrate how multiplex targeting by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 can be used to systematically elucidate the functions of multigene families.-Voloshanenko, O., Gmach, P., Winter, J., Kranz, D., Boutros, M. Mapping of Wnt-Frizzled interactions by multiplex CRISPR targeting of receptor gene families. © The Author(s).

CRISPR loci reveal networks of gene exchange in archaea.

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Brodt, Avital; Lurie-Weinberger, Mor N; Gophna, Uri

2011-12-21

CRISPR (Clustered, Regularly, Interspaced, Short, Palindromic Repeats) loci provide prokaryotes with an adaptive immunity against viruses and other mobile genetic elements. CRISPR arrays can be transcribed and processed into small crRNA molecules, which are then used by the cell to target the foreign nucleic acid. Since spacers are accumulated by active CRISPR/Cas systems, the sequences of these spacers provide a record of the past "infection history" of the organism. Here we analyzed all currently known spacers present in archaeal genomes and identified their source by DNA similarity. While nearly 50% of archaeal spacers matched mobile genetic elements, such as plasmids or viruses, several others matched chromosomal genes of other organisms, primarily other archaea. Thus, networks of gene exchange between archaeal species were revealed by the spacer analysis, including many cases of inter-genus and inter-species gene transfer events. Spacers that recognize viral sequences tend to be located further away from the leader sequence, implying that there exists a selective pressure for their retention. CRISPR spacers provide direct evidence for extensive gene exchange in archaea, especially within genera, and support the current dogma where the primary role of the CRISPR/Cas system is anti-viral and anti-plasmid defense. This article was reviewed by: Profs. W. Ford Doolittle, John van der Oost, Christa Schleper (nominated by board member Prof. J Peter Gogarten).

Application of CRISPR/Cas9 Genome Editing Technology for the Improvement of Crops Cultivated in Tropical Climates: Recent Progress, Prospects, and Challenges.

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Haque, Effi; Taniguchi, Hiroaki; Hassan, Md Mahmudul; Bhowmik, Pankaj; Karim, M Rezaul; Śmiech, Magdalena; Zhao, Kaijun; Rahman, Mahfuzur; Islam, Tofazzal

2018-01-01

The world population is expected to increase from 7.3 to 9.7 billion by 2050. Pest outbreak and increased abiotic stresses due to climate change pose a high risk to tropical crop production. Although conventional breeding techniques have significantly increased crop production and yield, new approaches are required to further improve crop production in order to meet the global growing demand for food. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 (CRISPR-associated protein9) genome editing technology has shown great promise for quickly addressing emerging challenges in agriculture. It can be used to precisely modify genome sequence of any organism including plants to achieve the desired trait. Compared to other genome editing tools such as zinc finger nucleases (ZFNs) and transcriptional activator-like effector nucleases (TALENs), CRISPR/Cas9 is faster, cheaper, precise and highly efficient in editing genomes even at the multiplex level. Application of CRISPR/Cas9 technology in editing the plant genome is emerging rapidly. The CRISPR/Cas9 is becoming a user-friendly tool for development of non-transgenic genome edited crop plants to counteract harmful effects from climate change and ensure future food security of increasing population in tropical countries. This review updates current knowledge and potentials of CRISPR/Cas9 for improvement of crops cultivated in tropical climates to gain resiliency against emerging pests and abiotic stresses.

Comparative genomics analysis of Streptococcus agalactiae reveals that isolates from cultured tilapia in China are closely related to the human strain A909.

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Liu, Guangjin; Zhang, Wei; Lu, Chengping

2013-11-11

Streptococcus agalactiae, also referred to as Group B Streptococcus (GBS), is a frequent resident of the rectovaginal tract in humans, and a major cause of neonatal infection. In addition, S. agalactiae is a known fish pathogen, which compromises food safety and represents a zoonotic hazard. The complete genome sequence of the piscine S. agalactiae isolate GD201008-001 was compared with 14 other piscine, human and bovine strains to explore their virulence determinants, evolutionary relationships and the genetic basis of host tropism in S. agalactiae. The pan-genome of S. agalactiae is open and its size increases with the addition of newly sequenced genomes. The core genes shared by all isolates account for 50 ~ 70% of any single genome. The Chinese piscine isolates GD201008-001 and ZQ0910 are phylogenetically distinct from the Latin American piscine isolates SA20-06 and STIR-CD-17, but are closely related to the human strain A909, in the context of the clustered regularly interspaced short palindromic repeats (CRISPRs), prophage, virulence-associated genes and phylogenetic relationships. We identified a unique 10 kb gene locus in Chinese piscine strains. Isolates from cultured tilapia in China have a close genomic relationship with the human strain A909. Our findings provide insight into the pathogenesis and host-associated genome content of piscine S. agalactiae isolated in China.

[Report of Relapse Typhoid Fever Cases from Kolkata, India: Recrudescence or Reinfection?

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Samajpati, Sriparna; Das, Surojit; Ray, Ujjwayini; Dutta, Shanta

2018-05-24

Three relapse cases were reported out of 107 hospital-attending typhoid cases within a period of 2 years (2014-2016) from Apollo Gleneagles Hospital, Kolkata, India. During the first episode of typhoid fever, 2 of the 3 cases were treated with ceftriaxone (CRO) for 7 days, and 1 was treated for 14 days. Six Salmonella Typhi (S. Typhi) isolates, obtained from the 3 patients during both typhoid episodes, were subjected to antimicrobial susceptibility testing, detection of quinolone resistance-determining region (QRDR) mutation and molecular subtyping by pulsed-field gel electrophoresis (PFGE), multiple-locus variable number tandem repeat analysis (MLVA), multilocus sequence typing (MLST), clustered regularly interspaced short palindromic repeats (CRISPR), and H58 haplotyping. Pairs of the S. Typhi strains isolated from two of the patients during the 1st and 2nd episodes were similar with respect to the antimicrobial resistance (AMR) profiles, QRDR mutations, and molecular subtypes; whereas, the S. Typhi strain pair isolated from the 3rd patient were different in their AMR profiles, QRDR mutations, and MLVA profiles. From these observations, it may be concluded that in spite of treating typhoid cases with CRO for 7-14 days, relapse of typhoid fever might occur. The article also showed the advantage of MLVA typing over PFGE, MLST, and CRISPR typing for the discrimination of strains isolated from the same patient in case of relapse of typhoid fever.

CRISPR/Cas9-Mediated Genomic Deletion of the Beta-1, 4 N-acetylgalactosaminyltransferase 1 Gene in Murine P19 Embryonal Carcinoma Cells Results in Low Sensitivity to Botulinum Neurotoxin Type C.

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Kentaro Tsukamoto

Full Text Available Botulinum neurotoxins produced by Clostridium botulinum cause flaccid paralysis by inhibiting neurotransmitter release at peripheral nerve terminals. Previously, we found that neurons derived from the murine P19 embryonal carcinoma cell line exhibited high sensitivity to botulinum neurotoxin type C. In order to prove the utility of P19 cells for the study of the intracellular mechanism of botulinum neurotoxins, ganglioside-knockout neurons were generated by deletion of the gene encoding beta-1,4 N-acetylgalactosaminyltransferase 1 in P19 cells using the clustered regularly interspaced short palindromic repeats combined with Cas9 (CRISPR/Cas9 system. By using this system, knockout cells could be generated more easily than with previous methods. The sensitivity of the generated beta-1,4 N-acetylgalactosaminyltransferase 1-depleted P19 neurons to botulinum neurotoxin type C was decreased considerably, and the exogenous addition of the gangliosides GD1a, GD1b, and GT1b restored the susceptibility of P19 cells to botulinum neurotoxin type C. In particular, addition of a mixture of these three ganglioside more effectively recovered the sensitivity of knockout cells compared to independent addition of GD1a, GD1b, or GT1b. Consequently, the genome-edited P19 cells generated by the CRISPR/Cas9 system were useful for identifying and defining the intracellular molecules involved in the toxic action of botulinum neurotoxins.

The Application of NHEJ-CRISPR/Cas9 and Cre-Lox System in the Generation of Bivalent Duck Enteritis Virus Vaccine against Avian Influenza Virus

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Pengxiang Chang

2018-02-01

Full Text Available Duck-targeted vaccines to protect against avian influenza are critically needed to aid in influenza disease control efforts in regions where ducks are endemic for highly pathogenic avian influenza (HPAI. Duck enteritis virus (DEV is a promising candidate viral vector for development of vaccines targeting ducks, owing to its large genome and narrow host range. The clustered regularly interspaced palindromic repeats (CRISPR/Cas9 system is a versatile gene-editing tool that has proven beneficial for gene modification and construction of recombinant DNA viral vectored vaccines. Currently, there are two commonly used methods for gene insertion: non-homologous end-joining (NHEJ and homology-directed repair (HDR. Owing to its advantages in efficiency and independence from molecular requirements of the homologous arms, we utilized NHEJ-dependent CRISPR/Cas9 to insert the influenza hemagglutinin (HA antigen expression cassette into the DEV genome. The insert was initially tagged with reporter green fluorescence protein (GFP, and a Cre-Lox system was later used to remove the GFP gene insert. Furthermore, a universal donor plasmid system was established by introducing double bait sequences that were independent of the viral genome. In summary, we provide proof of principle for generating recombinant DEV viral vectored vaccines against the influenza virus using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

Efficient and Heritable Gene Targeting in Tilapia by CRISPR/Cas9

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Li, Minghui; Yang, Huihui; Zhao, Jiue; Fang, Lingling; Shi, Hongjuan; Li, Mengru; Sun, Yunlv; Zhang, Xianbo; Jiang, Dongneng; Zhou, Linyan; Wang, Deshou

2014-01-01

Studies of gene function in non-model animals have been limited by the approaches available for eliminating gene function. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated) system has recently become a powerful tool for targeted genome editing. Here, we report the use of the CRISPR/Cas9 system to disrupt selected genes, including nanos2, nanos3, dmrt1, and foxl2, with efficiencies as high as 95%. In addition, mutations in dmrt1 and foxl2 induced by CRISPR/Cas9 were efficiently transmitted through the germline to F1. Obvious phenotypes were observed in the G0 generation after mutation of germ cell or somatic cell-specific genes. For example, loss of Nanos2 and Nanos3 in XY and XX fish resulted in germ cell-deficient gonads as demonstrated by GFP labeling and Vasa staining, respectively, while masculinization of somatic cells in both XY and XX gonads was demonstrated by Dmrt1 and Cyp11b2 immunohistochemistry and by up-regulation of serum androgen levels. Our data demonstrate that targeted, heritable gene editing can be achieved in tilapia, providing a convenient and effective approach for generating loss-of-function mutants. Furthermore, our study shows the utility of the CRISPR/Cas9 system for genetic engineering in non-model species like tilapia and potentially in many other teleost species. PMID:24709635

Efficient CRISPR/Cas9-Based Genome Engineering in Human Pluripotent Stem Cells.

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Kime, Cody; Mandegar, Mohammad A; Srivastava, Deepak; Yamanaka, Shinya; Conklin, Bruce R; Rand, Tim A

2016-01-01

Human pluripotent stem cells (hPS cells) are rapidly emerging as a powerful tool for biomedical discovery. The advent of human induced pluripotent stem cells (hiPS cells) with human embryonic stem (hES)-cell-like properties has led to hPS cells with disease-specific genetic backgrounds for in vitro disease modeling and drug discovery as well as mechanistic and developmental studies. To fully realize this potential, it will be necessary to modify the genome of hPS cells with precision and flexibility. Pioneering experiments utilizing site-specific double-strand break (DSB)-mediated genome engineering tools, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have paved the way to genome engineering in previously recalcitrant systems such as hPS cells. However, these methods are technically cumbersome and require significant expertise, which has limited adoption. A major recent advance involving the clustered regularly interspaced short palindromic repeats (CRISPR) endonuclease has dramatically simplified the effort required for genome engineering and will likely be adopted widely as the most rapid and flexible system for genome editing in hPS cells. In this unit, we describe commonly practiced methods for CRISPR endonuclease genomic editing of hPS cells into cell lines containing genomes altered by insertion/deletion (indel) mutagenesis or insertion of recombinant genomic DNA. Copyright © 2016 John Wiley & Sons, Inc.

CRISPR-Cas systems exploit viral DNA injection to establish and maintain adaptive immunity.

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Modell, Joshua W; Jiang, Wenyan; Marraffini, Luciano A

2017-04-06

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems provide protection against viral and plasmid infection by capturing short DNA sequences from these invaders and integrating them into the CRISPR locus of the prokaryotic host. These sequences, known as spacers, are transcribed into short CRISPR RNA guides that specify the cleavage site of Cas nucleases in the genome of the invader. It is not known when spacer sequences are acquired during viral infection. Here, to investigate this, we tracked spacer acquisition in Staphylococcus aureus cells harbouring a type II CRISPR-Cas9 system after infection with the staphylococcal bacteriophage ϕ12. We found that new spacers were acquired immediately after infection preferentially from the cos site, the viral free DNA end that is first injected into the cell. Analysis of spacer acquisition after infection with mutant phages demonstrated that most spacers are acquired during DNA injection, but not during other stages of the viral cycle that produce free DNA ends, such as DNA replication or packaging. Finally, we showed that spacers acquired from early-injected genomic regions, which direct Cas9 cleavage of the viral DNA immediately after infection, provide better immunity than spacers acquired from late-injected regions. Our results reveal that CRISPR-Cas systems exploit the phage life cycle to generate a pattern of spacer acquisition that ensures a successful CRISPR immune response.

CRISPRDetect: A flexible algorithm to define CRISPR arrays.

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Biswas, Ambarish; Staals, Raymond H J; Morales, Sergio E; Fineran, Peter C; Brown, Chris M

2016-05-17

CRISPR (clustered regularly interspaced short palindromic repeats) RNAs provide the specificity for noncoding RNA-guided adaptive immune defence systems in prokaryotes. CRISPR arrays consist of repeat sequences separated by specific spacer sequences. CRISPR arrays have previously been identified in a large proportion of prokaryotic genomes. However, currently available detection algorithms do not utilise recently discovered features regarding CRISPR loci. We have developed a new approach to automatically detect, predict and interactively refine CRISPR arrays. It is available as a web program and command line from bioanalysis.otago.ac.nz/CRISPRDetect. CRISPRDetect discovers putative arrays, extends the array by detecting additional variant repeats, corrects the direction of arrays, refines the repeat/spacer boundaries, and annotates different types of sequence variations (e.g. insertion/deletion) in near identical repeats. Due to these features, CRISPRDetect has significant advantages when compared to existing identification tools. As well as further support for small medium and large repeats, CRISPRDetect identified a class of arrays with 'extra-large' repeats in bacteria (repeats 44-50 nt). The CRISPRDetect output is integrated with other analysis tools. Notably, the predicted spacers can be directly utilised by CRISPRTarget to predict targets. CRISPRDetect enables more accurate detection of arrays and spacers and its gff output is suitable for inclusion in genome annotation pipelines and visualisation. It has been used to analyse all complete bacterial and archaeal reference genomes.

Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes.

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Wang, Bangmei; Li, Kunyu; Wang, Amy; Reiser, Michelle; Saunders, Thom; Lockey, Richard F; Wang, Jia-Wang

2015-10-01

The clustered regularly interspaced short palindromic repeat (CRISPR) gene editing technique, based on the non-homologous end-joining (NHEJ) repair pathway, has been used to generate gene knock-outs with variable sizes of small insertion/deletions with high efficiency. More precise genome editing, either the insertion or deletion of a desired fragment, can be done by combining the homology-directed-repair (HDR) pathway with CRISPR cleavage. However, HDR-mediated gene knock-in experiments are typically inefficient, and there have been no reports of successful gene knock-in with DNA fragments larger than 4 kb. Here, we describe the targeted insertion of large DNA fragments (7.4 and 5.8 kb) into the genomes of mouse embryonic stem (ES) cells and zygotes, respectively, using the CRISPR/HDR technique without NHEJ inhibitors. Our data show that CRISPR/HDR without NHEJ inhibitors can result in highly efficient gene knock-in, equivalent to CRISPR/HDR with NHEJ inhibitors. Although NHEJ is the dominant repair pathway associated with CRISPR-mediated double-strand breaks (DSBs), and biallelic gene knock-ins are common, NHEJ and biallelic gene knock-ins were not detected. Our results demonstrate that efficient targeted insertion of large DNA fragments without NHEJ inhibitors is possible, a result that should stimulate interest in understanding the mechanisms of high efficiency CRISPR targeting in general.

[Detection of CRISPR and its relationship to drug resistance in Shigella].

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Wang, Linlin; Wang, Yingfang; Duan, Guangcai; Xue, Zerun; Guo, Xiangjiao; Wang, Pengfei; Xi, Yuanlin; Yang, Haiyan

2015-04-04

To detect clustered regularly interspaced short palindromic repeats (CRISPR) in Shigella, and to analyze its relationship to drug resistance. Four pairs of primers were used for the detection of convincing CRISPR structures CRISPR-S2 and CRISPR-S4, questionable CRISPR structures CRISPR-S1 and CRISPR-S3 in 60 Shigella strains. All primers were designed using sequences in CRISPR database. CRISPR Finder was used to analyze CRISPR and susceptibilities of Shigella strains were tested by agar diffusion method. Furthermore, we analyzed the relationship between drug resistance and CRISPR-S4. The positive rate of convincing CRISPR structures was 95%. The four CRISPR loci formed 12 spectral patterns (A-L), all of which contained convincing CRISPR structures except type K. We found one new repeat and 12 new spacers. The multi-drug resistance rate was 53. 33% . We found no significant difference between CRISPR-S4 and drug resistant. However, the repeat sequence of CRISPR-S4 in multi- or TE-resistance strains was mainly R4.1 with AC deletions in the 3' end, and the spacer sequences of CRISPR-S4 in multi-drug resistance strains were mainly Sp5.1, Sp6.1 and Sp7. CRISPR was common in Shigella. Variations df repeat sequences and diversities of spacer sequences might be related to drug resistance in Shigella.

[Changes of resistant phenotype and CRISPR/Cas system of four Shigella strains passaged for 90 times without antibiotics].

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Zhang, B; Hong, L J; Duan, G C; Liang, W J; Yang, H Y; Xi, Y L

2017-02-10

Objective: To explore the stability of resistant phenotypes and changes of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) gene system on four Shigella strains in the absence of antibiotics. Methods: Four clinical isolated Shigella strains that resistant to different antibiotics were consecutive passaged for 90 times without antibiotics. Agar dilution method was used to determine the minimum inhibitory concentration of Shigella strains. After sequence analysis with PCR, CRISPR Finder and Clustal X 2.1 were applied to identify the changes of CRISPR loci in the Shigella strains. Results: After the consecutive transfer of 90 generations, sensitivity to certain antibiotics of four Shigella strains with different drug resistant spectrums increased. Mel-sf1998024/zz resistance to ampicillin, cephalexin, cefotaxime, chloramphenicol decreased, mel-s2014026/sx resistance to norfloxacin, trimethoprim decreased, mel-sf2004004/sx drug resistance to ampicillin, cefuroxime, cefotaxime, chloramphenicol, trimethoprim decreased and mel-sf2013004/bj resistance to chloramphenicol decreased. The spacer of which matched gene codes Cas and its upstream repeat in 3'end of CRISPR3 got lost in mel-sf1998024/zz and mel-sf2013004/bj. Conclusions: Shigella strains could reduce or lose their resistance to some antibiotics after consecutive transfers, without the interference of antibiotics. CRISPR3 locus had dynamic spacers in Shigella strains while CRISPR3 locus and cas genes might have been co-evolved.

A comprehensive overview of computational resources to aid in precision genome editing with engineered nucleases.

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Periwal, Vinita

2017-07-01

Genome editing with engineered nucleases (zinc finger nucleases, TAL effector nucleases s and Clustered regularly inter-spaced short palindromic repeats/CRISPR-associated) has recently been shown to have great promise in a variety of therapeutic and biotechnological applications. However, their exploitation in genetic analysis and clinical settings largely depends on their specificity for the intended genomic target. Large and complex genomes often contain highly homologous/repetitive sequences, which limits the specificity of genome editing tools and could result in off-target activity. Over the past few years, various computational approaches have been developed to assist the design process and predict/reduce the off-target activity of these nucleases. These tools could be efficiently used to guide the design of constructs for engineered nucleases and evaluate results after genome editing. This review provides a comprehensive overview of various databases, tools, web servers and resources for genome editing and compares their features and functionalities. Additionally, it also describes tools that have been developed to analyse post-genome editing results. The article also discusses important design parameters that could be considered while designing these nucleases. This review is intended to be a quick reference guide for experimentalists as well as computational biologists working in the field of genome editing with engineered nucleases. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Improved traceability of Shiga-toxin-producing Escherichia coli using CRISPRs for detection and typing.

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Delannoy, Sabine; Beutin, Lothar; Fach, Patrick

2016-05-01

Among strains of Shiga-toxin-producing Escherichia coli (STEC), seven serogroups (O26, O45, O103, O111, O121, O145, and O157) are frequently associated with severe clinical illness in humans. The development of methods for their reliable detection from complex samples such as food has been challenging thus far, and is currently based on the PCR detection of the major virulence genes stx1, stx2, and eae, and O-serogroup-specific genes. However, this approach lacks resolution. Moreover, new STEC serotypes are continuously emerging worldwide. For example, in May 2011, strains belonging to the hitherto rarely detected STEC serotype O104:H4 were identified as causative agents of one of the world's largest outbreak of disease with a high incidence of hemorrhagic colitis and hemolytic uremic syndrome in the infected patients. Discriminant typing of pathogens is crucial for epidemiological surveillance and investigations of outbreaks, and especially for tracking and tracing in case of accidental and deliberate contamination of food and water samples. Clustered regularly interspaced short palindromic repeats (CRISPRs) are composed of short, highly conserved DNA repeats separated by unique sequences of similar length. This distinctive sequence signature of CRISPRs can be used for strain typing in several bacterial species including STEC. This review discusses how CRISPRs have recently been used for STEC identification and typing.

Engineering Plant Immunity: Using CRISPR/Cas9 to Generate Virus Resistance

KAUST Repository

Zaidi, Syed Shan-e-Ali; Tashkandi, Manal; Mansoor, Shahid; Mahfouz, Magdy M.

2016-01-01

Plant viruses infect many economically important crops, including wheat, cotton, maize, cassava, and other vegetables. These viruses pose a serious threat to agriculture worldwide, as decreases in cropland area per capita may cause production to fall short of that required to feed the increasing world population. Under these circumstances, conventional strategies can fail to control rapidly evolving and emerging plant viruses. Genome-engineering strategies have recently emerged as promising tools to introduce desirable traits in many eukaryotic species, including plants. Among these genome engineering technologies, the CRISPR (clustered regularly interspaced palindromic repeats)/CRISPR-associated 9 (CRISPR/Cas9) system has received special interest because of its simplicity, efficiency, and reproducibility. Recent studies have used CRISPR/Cas9 to engineer virus resistance in plants, either by directly targeting and cleaving the viral genome, or by modifying the host plant genome to introduce viral immunity. Here, we briefly describe the biology of the CRISPR/Cas9 system and plant viruses, and how different genome engineering technologies have been used to target these viruses. We further describe the main findings from recent studies of CRISPR/Cas9-mediated viral interference and discuss how these findings can be applied to improve global agriculture. We conclude by pinpointing the gaps in our knowledge and the outstanding questions regarding CRISPR/Cas9-mediated viral immunity.

Improved image quality of cone beam CT scans for radiotherapy image guidance using fiber-interspaced antiscatter grid

International Nuclear Information System (INIS)

Stankovic, Uros; Herk, Marcel van; Ploeger, Lennert S.; Sonke, Jan-Jakob

2014-01-01

Purpose: Medical linear accelerator mounted cone beam CT (CBCT) scanner provides useful soft tissue contrast for purposes of image guidance in radiotherapy. The presence of extensive scattered radiation has a negative effect on soft tissue visibility and uniformity of CBCT scans. Antiscatter grids (ASG) are used in the field of diagnostic radiography to mitigate the scatter. They usually do increase the contrast of the scan, but simultaneously increase the noise. Therefore, and considering other scatter mitigation mechanisms present in a CBCT scanner, the applicability of ASGs with aluminum interspacing for a wide range of imaging conditions has been inconclusive in previous studies. In recent years, grids using fiber interspacers have appeared, providing grids with higher scatter rejection while maintaining reasonable transmission of primary radiation. The purpose of this study was to evaluate the impact of one such grid on CBCT image quality. Methods: The grid used (Philips Medical Systems) had ratio of 21:1, frequency 36 lp/cm, and nominal selectivity of 11.9. It was mounted on the kV flat panel detector of an Elekta Synergy linear accelerator and tested in a phantom and a clinical study. Due to the flex of the linac and presence of gridline artifacts an angle dependent gain correction algorithm was devised to mitigate resulting artifacts. Scan reconstruction was performed using XVI4.5 augmented with inhouse developed image lag correction and Hounsfield unit calibration. To determine the necessary parameters for Hounsfield unit calibration and software scatter correction parameters, the Catphan 600 (The Phantom Laboratory) phantom was used. Image quality parameters were evaluated using CIRS CBCT Image Quality and Electron Density Phantom (CIRS) in two different geometries: one modeling head and neck and other pelvic region. Phantoms were acquired with and without the grid and reconstructed with and without software correction which was adapted for the different

A guild of 45 CRISPR-associated (Cas protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes.

Directory of Open Access Journals (Sweden)

Daniel H Haft

2005-11-01

Full Text Available Clustered regularly interspaced short palindromic repeats (CRISPRs are a family of DNA direct repeats found in many prokaryotic genomes. Repeats of 21-37 bp typically show weak dyad symmetry and are separated by regularly sized, nonrepetitive spacer sequences. Four CRISPR-associated (Cas protein families, designated Cas1 to Cas4, are strictly associated with CRISPR elements and always occur near a repeat cluster. Some spacers originate from mobile genetic elements and are thought to confer "immunity" against the elements that harbor these sequences. In the present study, we have systematically investigated uncharacterized proteins encoded in the vicinity of these CRISPRs and found many additional protein families that are strictly associated with CRISPR loci across multiple prokaryotic species. Multiple sequence alignments and hidden Markov models have been built for 45 Cas protein families. These models identify family members with high sensitivity and selectivity and classify key regulators of development, DevR and DevS, in Myxococcus xanthus as Cas proteins. These identifications show that CRISPR/cas gene regions can be quite large, with up to 20 different, tandem-arranged cas genes next to a repeat cluster or filling the region between two repeat clusters. Distinctive subsets of the collection of Cas proteins recur in phylogenetically distant species and correlate with characteristic repeat periodicity. The analyses presented here support initial proposals of mobility of these units, along with the likelihood that loci of different subtypes interact with one another as well as with host cell defensive, replicative, and regulatory systems. It is evident from this analysis that CRISPR/cas loci are larger, more complex, and more heterogeneous than previously appreciated.

Distance-regular graphs

NARCIS (Netherlands)

van Dam, Edwin R.; Koolen, Jack H.; Tanaka, Hajime

2016-01-01

This is a survey of distance-regular graphs. We present an introduction to distance-regular graphs for the reader who is unfamiliar with the subject, and then give an overview of some developments in the area of distance-regular graphs since the monograph 'BCN'[Brouwer, A.E., Cohen, A.M., Neumaier,

Regular expressions cookbook

CERN Document Server

Goyvaerts, Jan

2009-01-01

This cookbook provides more than 100 recipes to help you crunch data and manipulate text with regular expressions. Every programmer can find uses for regular expressions, but their power doesn't come worry-free. Even seasoned users often suffer from poor performance, false positives, false negatives, or perplexing bugs. Regular Expressions Cookbook offers step-by-step instructions for some of the most common tasks involving this tool, with recipes for C#, Java, JavaScript, Perl, PHP, Python, Ruby, and VB.NET. With this book, you will: Understand the basics of regular expressions through a

LL-regular grammars

NARCIS (Netherlands)

Nijholt, Antinus

1980-01-01

Culik II and Cogen introduced the class of LR-regular grammars, an extension of the LR(k) grammars. In this paper we consider an analogous extension of the LL(k) grammars called the LL-regular grammars. The relation of this class of grammars to other classes of grammars will be shown. Any LL-regular

Numerical taxonomy of the genus Pestivirus: new software for genotyping based on the palindromic nucleotide substitutions method.

Science.gov (United States)

Giangaspero, Massimo; Apicella, Claudio; Harasawa, Ryô

2013-09-01

The genus Pestivirus from the family Flaviviridae is represented by four established species; Bovine viral diarrhea virus 1 (BVDV-1); Bovine viral diarrhea virus 2 (BVDV-2); Border disease virus (BDV); and Classical swine fever virus (CSFV); as well a tentative species from a Giraffe. The palindromic nucleotide substitutions (PNS) in the 5' untranslated region (UTR) of Pestivirus RNA has been described as a new, simple and practical method for genotyping. New software is described, also named PNS, that was prepared specifically for this PNS genotyping procedure. Pestivirus identification using PNS was evaluated on five hundred and forty-three sequences at genus, species and genotype level using this software. The software is freely available at www.pns-software.com. Copyright © 2013 Elsevier B.V. All rights reserved.

The dyad palindromic glutathione transferase P enhancer binds multiple factors including AP1.

Science.gov (United States)

Diccianni, M B; Imagawa, M; Muramatsu, M

1992-10-11

Glutathione Transferase P (GST-P) gene expression is dominantly regulated by an upstream enhancer (GPEI) consisting of a dyad of palindromically oriented imperfect TPA (12-O-tetradecanoyl-phorbol-13-acetate)-responsive elements (TRE). GPEI is active in AP1-lacking F9 cells as well in AP1-containing HeLa cells. Despite GPEI's similarity to a TRE, c-jun co-transfection has only a minimal effect on transactivation. Antisense c-jun and c-fos co-transfection experiments further demonstrate the lack of a role for AP1 in GPEI mediated trans-activation in F9 cells, although endogenously present AP1 can influence GPEI in HeLa cells. Co-transfection of delta fosB with c-jun, which forms an inactive c-Jun/delta FosB heterodimer that binds TRE sequences, inhibits GPEI-mediated transcription in AP1-lacking F9 cells as well as AP1-containing HeLa cells. These data suggest novel factor(s) other than AP1 are influencing GPEI. Binding studies reveal multiple nucleoproteins bind to GPEI. These factors are likely responsible for the high level of GPEI-mediated transcription observed in the absence of AP1 and during hepatocarcinogenesis.

The concurrent use of novel soil surface microclimate measurements to evaluate CO2 pulses in biocrusted interspaces in a cool desert ecosystem

Science.gov (United States)

Tucker, Colin; McHugh, Theresa A.; Howell, Armin; Gill, Richard; Weber, Bettina; Belnap, Jayne; Grote, Ed; Reed, Sasha C.

2017-01-01

Carbon cycling associated with biological soil crusts, which occupy interspaces between vascular plants in drylands globally, may be an important part of the coupled climate-carbon cycle of the Earth system. A major challenge to understanding CO2 fluxes in these systems is that much of the biotic and biogeochemical activity occurs in the upper few mm of the soil surface layer (i.e., the ‘mantle of fertility’), which exhibits highly dynamic and difficult to measure temperature and moisture fluctuations. Here, we report a multi-sensor approach to simultaneously measuring temperature and moisture of this biocrust surface layer (0–2 mm), and the deeper soil profile, concurrent with automated measurement of surface soil CO2effluxes. Our results illuminate robust relationships between biocrust water content and field CO2 pulses that have previously been difficult to detect and explain. All observed CO2 pulses over the measurement period corresponded to surface wetting events, including when the wetting events did not penetrate into the soil below the biocrust layer (0–2 mm). The variability of temperature and moisture of the biocrust surface layer was much greater than even in the 0–5 cm layer of the soil beneath the biocrust, or deeper in the soil profile. We therefore suggest that coupling surface measurements of biocrust moisture and temperature to automated CO2flux measurements may greatly improve our understanding of the climatic sensitivity of carbon cycling in biocrusted interspaces in our study region, and that this method may be globally relevant and applicable.

An iterative method for Tikhonov regularization with a general linear regularization operator

NARCIS (Netherlands)

Hochstenbach, M.E.; Reichel, L.

2010-01-01

Tikhonov regularization is one of the most popular approaches to solve discrete ill-posed problems with error-contaminated data. A regularization operator and a suitable value of a regularization parameter have to be chosen. This paper describes an iterative method, based on Golub-Kahan

Regular Expression Pocket Reference

CERN Document Server

Stubblebine, Tony

2007-01-01

This handy little book offers programmers a complete overview of the syntax and semantics of regular expressions that are at the heart of every text-processing application. Ideal as a quick reference, Regular Expression Pocket Reference covers the regular expression APIs for Perl 5.8, Ruby (including some upcoming 1.9 features), Java, PHP, .NET and C#, Python, vi, JavaScript, and the PCRE regular expression libraries. This concise and easy-to-use reference puts a very powerful tool for manipulating text and data right at your fingertips. Composed of a mixture of symbols and text, regular exp

Crystal optimization and preliminary diffraction data analysis of the Smad1 MH1 domain bound to a palindromic SBE DNA element

International Nuclear Information System (INIS)

Baburajendran, Nithya; Palasingam, Paaventhan; Ng, Calista Keow Leng; Jauch, Ralf; Kolatkar, Prasanna R.

2009-01-01

Crystals of palindromic SBE DNA-bound Smad1 MH1 domain diffracting to 2.7 Å resolution have been obtained. The bone morphogenetic protein (BMP) signalling pathway regulates diverse processes such as cell differentiation, anterior/posterior axis specification, cell growth and the formation of extra-embryonic tissues. The transcription factor Smad1 relays the BMP signal from the cytoplasm to the nucleus, where it binds short DNA-sequence motifs and regulates gene expression. However, how Smad1 selectively targets particular genomic regions is poorly understood. In order to understand the physical basis of the specific interaction of Smad1 with DNA and to contrast it with the highly homologous but functionally distinct Smad3 protein, the DNA-binding Mad-homology 1 (MH1) domain of Smad1 was cocrystallized with a 17-mer palindromic Smad-binding element (SBE). The extensive optimizations of the length, binding-site spacing and terminal sequences of the DNA element in combination with the other crystallization parameters necessary for obtaining diffraction-quality crystals are described here. A 2.7 Å resolution native data set was collected at the National Synchrotron Radiation Research Centre, Taiwan, from crystals grown in a solution containing 0.2 M ammonium tartrate dibasic, 20% PEG 3350, 3% 2-propanol and 10% glycerol. The data set was indexed and merged in space group P222, with unit-cell parameters a = 73.94, b = 77.49, c = 83.78 Å, α = β = γ = 90°. The solvent content in the unit cell is consistent with the presence of two Smad1 MH1 molecules bound to the duplex DNA in the asymmetric unit

CRISPR/Cas9 mediated high efficiency knockout of the eye color gene vermillion in Helicoverpa zea (Boddie)

Science.gov (United States)

Among various genome editing tools available for functional genomic studies, reagents based on clustered regularly interspersed palindromic repeats (CRISPR) have gained popularity due to ease and versatility. CRISPR reagents consists of ribonucleoprotein (RNP) complexes formed by combining guide RNA...

The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats

Directory of Open Access Journals (Sweden)

Vergnaud Gilles

2007-05-01

Full Text Available Abstract Background In Archeae and Bacteria, the repeated elements called CRISPRs for "clustered regularly interspaced short palindromic repeats" are believed to participate in the defence against viruses. Short sequences called spacers are stored in-between repeated elements. In the current model, motifs comprising spacers and repeats may target an invading DNA and lead to its degradation through a proposed mechanism similar to RNA interference. Analysis of intra-species polymorphism shows that new motifs (one spacer and one repeated element are added in a polarised fashion. Although their principal characteristics have been described, a lot remains to be discovered on the way CRISPRs are created and evolve. As new genome sequences become available it appears necessary to develop automated scanning tools to make available CRISPRs related information and to facilitate additional investigations. Description We have produced a program, CRISPRFinder, which identifies CRISPRs and extracts the repeated and unique sequences. Using this software, a database is constructed which is automatically updated monthly from newly released genome sequences. Additional tools were created to allow the alignment of flanking sequences in search for similarities between different loci and to build dictionaries of unique sequences. To date, almost six hundred CRISPRs have been identified in 475 published genomes. Two Archeae out of thirty-seven and about half of Bacteria do not possess a CRISPR. Fine analysis of repeated sequences strongly supports the current view that new motifs are added at one end of the CRISPR adjacent to the putative promoter. Conclusion It is hoped that availability of a public database, regularly updated and which can be queried on the web will help in further dissecting and understanding CRISPR structure and flanking sequences evolution. Subsequent analyses of the intra-species CRISPR polymorphism will be facilitated by CRISPRFinder and the

Multi-resistance strategy for viral diseases and short hairpin RNA verification method in pigs

Directory of Open Access Journals (Sweden)

Jong-nam Oh

2018-04-01

Full Text Available Objective Foot and mouth disease (FMD and porcine reproductive and respiratory syndrome (PRRS are major diseases that interrupt porcine production. Because they are viral diseases, vaccinations are of only limited effectiveness in preventing outbreaks. To establish an alternative multi-resistant strategy against FMD virus (FMDV and PRRS virus (PRRSV, the present study introduced two genetic modification techniques to porcine cells. Methods First, cluster of differentiation 163 (CD163, the PRRSV viral receptor, was edited with the clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 technique. The CD163 gene sequences of edited cells and control cells differed. Second, short hairpin RNA (shRNAs were integrated into the cells. The shRNAs, targeting the 3D gene of FMDV and the open reading frame 7 (ORF7 gene of PRRSV, were transferred into fibroblasts. We also developed an in vitro shRNA verification method with a target gene expression vector. Results shRNA activity was confirmed in vitro with vectors that expressed the 3D and ORF7 genes in the cells. Cells containing shRNAs showed lower transcript levels than cells with only the expression vectors. The shRNAs were integrated into CD163-edited cells to combine the two techniques, and the viral genes were suppressed in these cells. Conclusion We established a multi-resistant strategy against viral diseases and an in vitro shRNA verification method.

Two Novel Myoviruses from the North of Iraq Reveal Insights into Clostridium difficile Phage Diversity and Biology

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Srwa J. Rashid

2016-11-01

Full Text Available Bacteriophages (phages are increasingly being explored as therapeutic agents to combat bacterial diseases, including Clostridium difficile infections. Therapeutic phages need to be able to efficiently target and kill a wide range of clinically relevant strains. While many phage groups have yet to be investigated in detail, those with new and useful properties can potentially be identified when phages from newly studied geographies are characterised. Here, we report the isolation of C. difficile phages from soil samples from the north of Iraq. Two myoviruses, CDKM15 and CDKM9, were selected for detailed sequence analysis on the basis of their broad and potentially useful host range. CDKM9 infects 25/80 strains from 12/20 C. difficile ribotypes, and CDKM15 infects 20/80 strains from 9/20 ribotypes. Both phages can infect the clinically relevant ribotypes R027 and R001. Phylogenetic analysis based on whole genome sequencing revealed that the phages are genetically distinct from each other but closely related to other long-tailed myoviruses. A comparative genomic analysis revealed key differences in the genes predicted to encode for proteins involved in bacterial infection. Notably, CDKM15 carries a clustered regularly interspaced short palindromic repeat (CRISPR array with spacers that are homologous to sequences in the CDKM9 genome and of phages from diverse localities. The findings presented suggest a possible shared evolutionary past for these phages and provides evidence of their widespread dispersal.

Generation of a CRISPR database for Yersinia pseudotuberculosis complex and role of CRISPR-based immunity in conjugation.

Science.gov (United States)

Koskela, Katja A; Mattinen, Laura; Kalin-Mänttäri, Laura; Vergnaud, Gilles; Gorgé, Olivier; Nikkari, Simo; Skurnik, Mikael

2015-11-01

The clustered regularly interspaced short palindromic repeat - CRISPR-associated genes (CRISPR-Cas) system is used by bacteria and archaea against invading conjugative plasmids or bacteriophages. Central to this immunity system are genomic CRISPR loci that contain fragments of invading DNA. These are maintained as spacers in the CRISPR loci between direct repeats and the spacer composition in any bacterium reflects its evolutionary history. We analysed the CRISPR locus sequences of 335 Yersinia pseudotuberculosis complex strains. Altogether 1902 different spacer sequences were identified and these were used to generate a database for the spacer sequences. Only ∼10% of the spacer sequences found matching sequences. In addition, surprisingly few spacers were shared by Yersinia pestis and Y. pseudotuberculosis strains. Interestingly, 32 different protospacers were present in the conjugative plasmid pYptb32953. The corresponding spacers were identified from 35 different Y. pseudotuberculosis strains indicating that these strains had encountered pYptb32953 earlier. In conjugation experiments, pYptb32953-specific spacers generally prevented conjugation with spacer-positive and spacer-free strains. However, some strains with one to four spacers were invaded by pYptb32953 and some spacer-free strains were fully resistant. Also some spacer-positive strains were intermediate resistant to conjugation. This suggests that one or more other defence systems are determining conjugation efficiency independent of the CRISPR-Cas system. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Genetic Modification in Human Pluripotent Stem Cells by Homologous Recombination and CRISPR/Cas9 System.

Science.gov (United States)

Xue, Haipeng; Wu, Jianbo; Li, Shenglan; Rao, Mahendra S; Liu, Ying

2016-01-01

Genetic modification is an indispensable tool to study gene function in normal development and disease. The recent breakthrough of creating human induced pluripotent stem cells (iPSCs) by defined factors (Takahashi et al., Cell 131:861-872, 2007) provides a renewable source of patient autologous cells that not only retain identical genetic information but also give rise to many cell types of the body including neurons and glia. Meanwhile, the rapid advancement of genome modification tools such as gene targeting by homologous recombination (Capecchi, Nat Rev Genet 6:507-512, 2005) and genome editing tools such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system, TALENs (Transcription activator-like effector nucleases), and ZFNs (Zinc finger nucleases) (Wang et al., Cell 153:910-918, 2013; Mali et al., Science 339:823-826, 2013; Hwang et al., Nat Biotechnol 31:227-229, 2013; Friedland et al., Nat Methods 10(8):741-743, 2013; DiCarlo et al., Nucleic Acids Res 41:4336-4343, 2013; Cong et al., Science 339:819-823, 2013) has greatly accelerated the development of human genome manipulation at the molecular level. This chapter describes the protocols for making neural lineage reporter lines using homologous recombination and the CRISPR/Cas system-mediated genome editing, including construction of targeting vectors, guide RNAs, transfection into hPSCs, and selection and verification of successfully targeted clones. This method can be applied to various needs of hPSC genetic engineering at high efficiency and high reliability.

CRISPR/Cas9 Mediated Genome Engineering for Improvement of Horticultural Crops.

Science.gov (United States)

Karkute, Suhas G; Singh, Achuit K; Gupta, Om P; Singh, Prabhakar M; Singh, Bijendra

2017-01-01

Horticultural crops are an important part of agriculture for food as well as nutritional security. However, several pests and diseases along with adverse abiotic environmental factors pose a severe threat to these crops by affecting their quality and productivity. This warrants the effective and accelerated breeding programs by utilizing innovative biotechnological tools that can tackle aforementioned issues. The recent technique of genome editing by Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated 9 (CRISPR/Cas9) has greatly advanced the breeding for crop improvement due to its simplicity and high efficiency over other nucleases such as Zinc Finger Nucleases and Transcription Activator Like Effector Nucleases. CRISPR/Cas9 tool contains a non-specific Cas9 nuclease and a single guide RNA that directs Cas9 to the specific genomic location creating double-strand breaks and subsequent repair process creates insertion or deletion mutations. This is currently the widely adopted tool for reverse genetics, and crop improvement in large number of agricultural crops. The use of CRISPR/Cas9 in horticultural crops is limited to few crops due to lack of availability of regeneration protocols and sufficient sequence information in many horticultural crops. In this review, the present status of applicability of CRISPR/Cas9 in horticultural crops was discussed along with the challenges and future potential for possible improvement of these crops for their yield, quality, and resistance to biotic and abiotic stress.

CRISPR/Cas9-Advancing Orthopoxvirus Genome Editing for Vaccine and Vector Development.

Science.gov (United States)

Okoli, Arinze; Okeke, Malachy I; Tryland, Morten; Moens, Ugo

2018-01-22

The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (Cas9) technology is revolutionizing genome editing approaches. Its high efficiency, specificity, versatility, flexibility, simplicity and low cost have made the CRISPR/Cas9 system preferable to other guided site-specific nuclease-based systems such as TALENs (Transcription Activator-like Effector Nucleases) and ZFNs (Zinc Finger Nucleases) in genome editing of viruses. CRISPR/Cas9 is presently being applied in constructing viral mutants, preventing virus infections, eradicating proviral DNA, and inhibiting viral replication in infected cells. The successful adaptation of CRISPR/Cas9 to editing the genome of Vaccinia virus paves the way for its application in editing other vaccine/vector-relevant orthopoxvirus (OPXV) strains. Thus, CRISPR/Cas9 can be used to resolve some of the major hindrances to the development of OPXV-based recombinant vaccines and vectors, including sub-optimal immunogenicity; transgene and genome instability; reversion of attenuation; potential of spread of transgenes to wildtype strains and close contacts, which are important biosafety and risk assessment considerations. In this article, we review the published literature on the application of CRISPR/Cas9 in virus genome editing and discuss the potentials of CRISPR/Cas9 in advancing OPXV-based recombinant vaccines and vectors. We also discuss the application of CRISPR/Cas9 in combating viruses of clinical relevance, the limitations of CRISPR/Cas9 and the current strategies to overcome them.

CRISPR/Cas9: A Practical Approach in Date Palm Genome Editing

Directory of Open Access Journals (Sweden)

Muhammad N. Sattar

2017-08-01

Full Text Available The genetic modifications through breeding of crop plants have long been used to improve the yield and quality. However, precise genome editing (GE could be a very useful supplementary tool for improvement of crop plants by targeted genome modifications. Various GE techniques including ZFNs (zinc finger nucleases, TALENs (transcription activator-like effector nucleases, and most recently clustered regularly interspaced short palindromic repeats (CRISPR/Cas9 (CRISPR-associated protein 9-based approaches have been successfully employed for various crop plants including fruit trees. CRISPR/Cas9-based approaches hold great potential in GE due to their simplicity, competency, and versatility over other GE techniques. However, to the best of our knowledge no such genetic improvement has ever been developed in date palm—an important fruit crop in Oasis agriculture. The applications of CRISPR/Cas9 can be a challenging task in date palm GE due to its large and complex genome, high rate of heterozygosity and outcrossing, in vitro regeneration and screening of mutants, high frequency of single-nucleotide polymorphism in the genome and ultimately genetic instability. In this review, we addressed the potential application of CRISPR/Cas9-based approaches in date palm GE to improve the sustainable date palm production. The availability of the date palm whole genome sequence has made it feasible to use CRISPR/Cas9 GE approach for genetic improvement in this species. Moreover, the future prospects of GE application in date palm are also addressed in this review.

Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity.

Science.gov (United States)

Seamon, Kyle J; Light, Yooli K; Saada, Edwin A; Schoeniger, Joseph S; Harmon, Brooke

2018-06-05

The RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate its utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.

CRISPR/Cas9—Advancing Orthopoxvirus Genome Editing for Vaccine and Vector Development

Science.gov (United States)

Okoli, Arinze; Okeke, Malachy I.; Tryland, Morten; Moens, Ugo

2018-01-01

The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (Cas9) technology is revolutionizing genome editing approaches. Its high efficiency, specificity, versatility, flexibility, simplicity and low cost have made the CRISPR/Cas9 system preferable to other guided site-specific nuclease-based systems such as TALENs (Transcription Activator-like Effector Nucleases) and ZFNs (Zinc Finger Nucleases) in genome editing of viruses. CRISPR/Cas9 is presently being applied in constructing viral mutants, preventing virus infections, eradicating proviral DNA, and inhibiting viral replication in infected cells. The successful adaptation of CRISPR/Cas9 to editing the genome of Vaccinia virus paves the way for its application in editing other vaccine/vector-relevant orthopoxvirus (OPXV) strains. Thus, CRISPR/Cas9 can be used to resolve some of the major hindrances to the development of OPXV-based recombinant vaccines and vectors, including sub-optimal immunogenicity; transgene and genome instability; reversion of attenuation; potential of spread of transgenes to wildtype strains and close contacts, which are important biosafety and risk assessment considerations. In this article, we review the published literature on the application of CRISPR/Cas9 in virus genome editing and discuss the potentials of CRISPR/Cas9 in advancing OPXV-based recombinant vaccines and vectors. We also discuss the application of CRISPR/Cas9 in combating viruses of clinical relevance, the limitations of CRISPR/Cas9 and the current strategies to overcome them. PMID:29361752

Disabling Cas9 by an anti-CRISPR DNA mimic.

Science.gov (United States)

Shin, Jiyung; Jiang, Fuguo; Liu, Jun-Jie; Bray, Nicolas L; Rauch, Benjamin J; Baik, Seung Hyun; Nogales, Eva; Bondy-Denomy, Joseph; Corn, Jacob E; Doudna, Jennifer A

2017-07-01

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known, and the potential applications for Cas9 inhibitor proteins in mammalian cells have not been fully established. We show that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9-single-guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 Å resolution cryo-electron microscopy structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with a 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif. Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on preformed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.

High-Throughput Silencing Using the CRISPR-Cas9 System: A Review of the Benefits and Challenges.

Science.gov (United States)

Wade, Mark

2015-09-01

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has been seized upon with a fervor enjoyed previously by small interfering RNA (siRNA) and short hairpin RNA (shRNA) technologies and has enormous potential for high-throughput functional genomics studies. The decision to use this approach must be balanced with respect to adoption of existing platforms versus awaiting the development of more "mature" next-generation systems. Here, experience from siRNA and shRNA screening plays an important role, as issues such as targeting efficiency, pooling strategies, and off-target effects with those technologies are already framing debates in the CRISPR field. CRISPR/Cas can be exploited not only to knockout genes but also to up- or down-regulate gene transcription-in some cases in a multiplex fashion. This provides a powerful tool for studying the interaction among multiple signaling cascades in the same genetic background. Furthermore, the documented success of CRISPR/Cas-mediated gene correction (or the corollary, introduction of disease-specific mutations) provides proof of concept for the rapid generation of isogenic cell lines for high-throughput screening. In this review, the advantages and limitations of CRISPR/Cas are discussed and current and future applications are highlighted. It is envisaged that complementarities between CRISPR, siRNA, and shRNA will ensure that all three technologies remain critical to the success of future functional genomics projects. © 2015 Society for Laboratory Automation and Screening.

Determining the Specificity of Cascade Binding, Interference, and Primed Adaptation In Vivo in the Escherichia coli Type I-E CRISPR-Cas System

Directory of Open Access Journals (Sweden)

Lauren A. Cooper

2018-04-01

Full Text Available In clustered regularly interspaced short palindromic repeat (CRISPR-Cas (CRISPR-associated immunity systems, short CRISPR RNAs (crRNAs are bound by Cas proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptation in vivo, for the type I-E system of Escherichia coli. Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target. Consequently, a single crRNA promotes Cascade association with numerous off-target sites, and the endogenous E. coli crRNAs direct Cascade binding to >100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, >18 bp are required for both interference and primed adaptation. Hence, Cascade binding to suboptimal, off-target sites is inert. Our data support a model in which the initial Cascade association with DNA targets requires only limited sequence complementarity at the crRNA 5′ end whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and primed adaptation, requires extensive base pairing.

Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer.

Science.gov (United States)

Richter, Corinna; Dy, Ron L; McKenzie, Rebecca E; Watson, Bridget N J; Taylor, Corinda; Chang, James T; McNeil, Matthew B; Staals, Raymond H J; Fineran, Peter C

2014-07-01

Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼ 350 new spacers acquired in priming events and identified a 5'-protospacer-GG-3' protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2-3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

CRISPR loci reveal networks of gene exchange in archaea

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Brodt Avital

2011-12-01

Full Text Available Abstract Background CRISPR (Clustered, Regularly, Interspaced, Short, Palindromic Repeats loci provide prokaryotes with an adaptive immunity against viruses and other mobile genetic elements. CRISPR arrays can be transcribed and processed into small crRNA molecules, which are then used by the cell to target the foreign nucleic acid. Since spacers are accumulated by active CRISPR/Cas systems, the sequences of these spacers provide a record of the past "infection history" of the organism. Results Here we analyzed all currently known spacers present in archaeal genomes and identified their source by DNA similarity. While nearly 50% of archaeal spacers matched mobile genetic elements, such as plasmids or viruses, several others matched chromosomal genes of other organisms, primarily other archaea. Thus, networks of gene exchange between archaeal species were revealed by the spacer analysis, including many cases of inter-genus and inter-species gene transfer events. Spacers that recognize viral sequences tend to be located further away from the leader sequence, implying that there exists a selective pressure for their retention. Conclusions CRISPR spacers provide direct evidence for extensive gene exchange in archaea, especially within genera, and support the current dogma where the primary role of the CRISPR/Cas system is anti-viral and anti-plasmid defense. Open peer review This article was reviewed by: Profs. W. Ford Doolittle, John van der Oost, Christa Schleper (nominated by board member Prof. J Peter Gogarten

Interference activity of a minimal Type I CRISPR-Cas system from Shewanella putrefaciens.

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Dwarakanath, Srivatsa; Brenzinger, Susanne; Gleditzsch, Daniel; Plagens, André; Klingl, Andreas; Thormann, Kai; Randau, Lennart

2015-10-15

Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated) systems exist in bacterial and archaeal organisms and provide immunity against foreign DNA. The Cas protein content of the DNA interference complexes (termed Cascade) varies between different CRISPR-Cas subtypes. A minimal variant of the Type I-F system was identified in proteobacterial species including Shewanella putrefaciens CN-32. This variant lacks a large subunit (Csy1), Csy2 and Csy3 and contains two unclassified cas genes. The genome of S. putrefaciens CN-32 contains only five Cas proteins (Cas1, Cas3, Cas6f, Cas1821 and Cas1822) and a single CRISPR array with 81 spacers. RNA-Seq analyses revealed the transcription of this array and the maturation of crRNAs (CRISPR RNAs). Interference assays based on plasmid conjugation demonstrated that this CRISPR-Cas system is active in vivo and that activity is dependent on the recognition of the dinucleotide GG PAM (Protospacer Adjacent Motif) sequence and crRNA abundance. The deletion of cas1821 and cas1822 reduced the cellular crRNA pool. Recombinant Cas1821 was shown to form helical filaments bound to RNA molecules, which suggests its role as the Cascade backbone protein. A Cascade complex was isolated which contained multiple Cas1821 copies, Cas1822, Cas6f and mature crRNAs. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Massive activation of archaeal defense genes during viral infection.

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Quax, Tessa E F; Voet, Marleen; Sismeiro, Odile; Dillies, Marie-Agnes; Jagla, Bernd; Coppée, Jean-Yves; Sezonov, Guennadi; Forterre, Patrick; van der Oost, John; Lavigne, Rob; Prangishvili, David

2013-08-01

Archaeal viruses display unusually high genetic and morphological diversity. Studies of these viruses proved to be instrumental for the expansion of knowledge on viral diversity and evolution. The Sulfolobus islandicus rod-shaped virus 2 (SIRV2) is a model to study virus-host interactions in Archaea. It is a lytic virus that exploits a unique egress mechanism based on the formation of remarkable pyramidal structures on the host cell envelope. Using whole-transcriptome sequencing, we present here a global map defining host and viral gene expression during the infection cycle of SIRV2 in its hyperthermophilic host S. islandicus LAL14/1. This information was used, in combination with a yeast two-hybrid analysis of SIRV2 protein interactions, to advance current understanding of viral gene functions. As a consequence of SIRV2 infection, transcription of more than one-third of S. islandicus genes was differentially regulated. While expression of genes involved in cell division decreased, those genes playing a role in antiviral defense were activated on a large scale. Expression of genes belonging to toxin-antitoxin and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems was specifically pronounced. The observed different degree of activation of various CRISPR-Cas systems highlights the specialized functions they perform. The information on individual gene expression and activation of antiviral defense systems is expected to aid future studies aimed at detailed understanding of the functions and interplay of these systems in vivo.

Type II CRISPR/Cas9 approach in the oncological therapy.

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Biagioni, A; Chillà, A; Andreucci, E; Laurenzana, A; Margheri, F; Peppicelli, S; Del Rosso, M; Fibbi, G

2017-06-15

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a prokaryotic adaptable immune mechanism used by many bacteria and archaea to protect themselves from foreign nucleic acids. This complex system can recognize and cut non-self DNA in order to provide the prokaryotic organisms a strong defense against foreign viral or plasmid attacks and make the cell immune from further assaults. Today, it has been adapted to be used in vitro and in vivo in eukaryotic cells to perform a complete and highly selective gene knockout or a specific gene editing. The ease of use and the low cost are only two features that have made it very popular among the scientific community and the possibility to be used as a clinical treatment in several genetic derived pathologies has rapidly spread its fame worldwide. However, CRISPR is still not fully understood and many efforts need to be done in order to make it a real power tool for the human clinical treatment especially for oncological patients. Indeed, since cancer originates from non-lethal genetic disorders, CRISPR discovery fuels the hope to strike tumors on their roots. More than 4000 papers regarding CRISPR were published in the last ten years and only few of them take in count the possible applications in oncology. The purpose of this review is to clarify many problematics on the CRISPR usage and highlight its potential in oncological therapy.

CRISPR-Cas gene-editing reveals RsmA and RsmC act through FlhDC to repress the SdhE flavinylation factor and control motility and prodigiosin production in Serratia.

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Hampton, Hannah G; McNeil, Matthew B; Paterson, Thomas J; Ney, Blair; Williamson, Neil R; Easingwood, Richard A; Bostina, Mihnea; Salmond, George P C; Fineran, Peter C

2016-06-01

SdhE is required for the flavinylation and activation of succinate dehydrogenase and fumarate reductase (FRD). In addition, SdhE is conserved in proteobacteria (α, β and γ) and eukaryotes. Although the function of this recently characterized family of proteins has been determined, almost nothing is known about how their genes are regulated. Here, the RsmA (CsrA) and RsmC (HexY) post-transcriptional and post-translational regulators have been identified and shown to repress sdhEygfX expression in Serratia sp. ATCC 39006. Conversely, the flagella master regulator complex, FlhDC, activated sdhEygfX transcription. To investigate the hierarchy of control, we developed a novel approach that utilized endogenous CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated) genome-editing by a type I-F system to generate a chromosomal point mutation in flhC. Mutation of flhC alleviated the ability of RsmC to repress sdhEygfX expression, whereas RsmA acted in both an FlhDC-dependent and -independent manner to inhibit sdhEygfX. Mutation of rsmA or rsmC, or overexpression of FlhDC, led to increased prodigiosin, biosurfactant, swimming and swarming. Consistent with the modulation of sdhE by motility regulators, we have demonstrated that SdhE and FRD are required for maximal flagella-dependent swimming. Together, these results demonstrate that regulators of both metabolism and motility (RsmA, RsmC and FlhDC) control the transcription of the sdhEygfX operon.

Cas9 in Genetically Modified Food Is Unlikely to Cause Food Allergy.

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Nakajima, Osamu; Nishimaki-Mogami, Tomoko; Kondo, Kazunari

2016-01-01

Genome editing has undergone rapid development during the last three years. It is anticipated that genetically modified organisms (GMOs) for food purposes will be widely produced using the clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR)/Cas9 system in the near future. However, the Cas9 gene may then enter the genomes of GMOs for food if the breeding process is not strictly managed, which could lead to the Cas9 protein or associated peptides being produced within these organisms. A variety of peptides could theoretically be produced from the Cas9 gene by using open reading frames different from that of Cas9 in the GMOs. In this study, Cas9 and the peptides potentially encoded by Cas9 genes were studied regarding their immunogenicity, in terms of the digestibility of Cas9 and the homology of the peptides to food allergens. First, the digestibility and thermal stability of Cas9 were studied. Digestibility was tested with natural or heat-denatured Cas9 in simulated gastric fluid in vitro. The two types of Cas9 were digested rapidly. Cas9 was also gradually degraded during heat treatment. Second, the peptides potentially encoded by Cas9 genes were examined for their homology to food allergens. Specifically, an 8-mer exact match search and a sliding 80-mer window search were performed using allergen databases. One of the peptides was found to have homology with a food allergen.

CRISPR associated diversity within a population of Sulfolobus islandicus.

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Nicole L Held

2010-09-01

Full Text Available Predator-prey models for virus-host interactions predict that viruses will cause oscillations of microbial host densities due to an arms race between resistance and virulence. A new form of microbial resistance, CRISPRs (clustered regularly interspaced short palindromic repeats are a rapidly evolving, sequence-specific immunity mechanism in which a short piece of invading viral DNA is inserted into the host's chromosome, thereby rendering the host resistant to further infection. Few studies have linked this form of resistance to population dynamics in natural microbial populations.We examined sequence diversity in 39 strains of the archeaon Sulfolobus islandicus from a single, isolated hot spring from Kamchatka, Russia to determine the effects of CRISPR immunity on microbial population dynamics. First, multiple housekeeping genetic markers identify a large clonal group of identical genotypes coexisting with a diverse set of rare genotypes. Second, the sequence-specific CRISPR spacer arrays split the large group of isolates into two very different groups and reveal extensive diversity and no evidence for dominance of a single clone within the population.The evenness of resistance genotypes found within this population of S. islandicus is indicative of a lack of strain dominance, in contrast to the prediction for a resistant strain in a simple predator-prey interaction. Based on evidence for the independent acquisition of resistant sequences, we hypothesize that CRISPR mediated clonal interference between resistant strains promotes and maintains diversity in this natural population.

Efficient Genome Editing in Chicken DF-1 Cells Using the CRISPR/Cas9 System

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Yichun Bai

2016-04-01

Full Text Available In recent years, genome engineering technology has provided unprecedented opportunities for site-specific modification of biological genomes. Clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR-associated (Cas 9 is one such means that can target a specific genome locus. It has been applied in human cells and many other organisms. Meanwhile, to efficiently enrich targeted cells, several surrogate systems have also been developed. However, very limited information exists on the application of CRISPR/Cas9 in chickens. In this study, we employed the CRISPR/Cas9 system to induce mutations in the peroxisome proliferator-activated receptor-γ (PPAR-γ, ATP synthase epsilon subunit (ATP5E, and ovalbumin (OVA genes in chicken DF-1 cells. The results of T7E1 assays showed that the mutation rate at the three different loci was 0.75%, 0.5%, and 3.0%, respectively. In order to improve the mutation efficiency, we used the PuroR gene for efficient enrichment of genetically modified cells with the surrogate reporter system. The mutation rate, as assessed via the T7E1 assay, increased to 60.7%, 61.3%, and 47.3%, and subsequent sequence analysis showed that the mutation efficiency increased to 94.7%, 95%, and 95%, respectively. In addition, there were no detectable off-target mutations in three potential off-target sites using the T7E1 assay. As noted above, the CRISPR/Cas9 system is a robust tool for chicken genome editing.

Predominant Expression of Hybrid N-Glycans Has Distinct Cellular Roles Relative to Complex and Oligomannose N-Glycans

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M. Kristen Hall

2016-06-01

Full Text Available Glycosylation modulates growth, maintenance, and stress signaling processes. Consequently, altered N-glycosylation is associated with reduced fitness and disease. Therefore, expanding our understanding of N-glycans in altering biological processes is of utmost interest. Herein, clustered regularly interspaced short palindromic repeats/caspase9 (CRISPR/Cas9 technology was employed to engineer a glycosylation mutant Chinese Hamster Ovary (CHO cell line, K16, which expresses predominantly hybrid type N-glycans. This newly engineered cell line enabled us to compare N-glycan effects on cellular properties of hybrid type N-glycans, to the well-established Pro−5 and Lec1 cell lines, which express complex and oligomannose types of N-glycans, respectively. Lectin binding studies revealed the predominant N-glycan expressed in K16 is hybrid type. Cell dissociation and migration assays demonstrated the greatest strength of cell–cell adhesion and fastest migratory rates for oligomannose N-glycans, and these properties decreased as oligomannose type were converted to hybrid type, and further decreased upon conversion to complex type. Next, we examined the roles of three general types of N-glycans on ectopic expression of E-cadherin, a cell–cell adhesion protein. Microscopy revealed more functional E-cadherin at the cell–cell border when N-glycans were oligomannose and these levels decreased as the oligomannose N-glycans were processed to hybrid and then to complex. Thus, we provide evidence that all three general types of N-glycans impact plasma membrane architecture and cellular properties.

RNA Interference in the Age of CRISPR: Will CRISPR Interfere with RNAi?

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Unnikrishnan Unniyampurath

2016-02-01

Full Text Available The recent emergence of multiple technologies for modifying gene structure has revolutionized mammalian biomedical research and enhanced the promises of gene therapy. Over the past decade, RNA interference (RNAi based technologies widely dominated various research applications involving experimental modulation of gene expression at the post-transcriptional level. Recently, a new gene editing technology, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR and the CRISPR-associated protein 9 (Cas9 (CRISPR/Cas9 system, has received unprecedented acceptance in the scientific community for a variety of genetic applications. Unlike RNAi, the CRISPR/Cas9 system is bestowed with the ability to introduce heritable precision insertions and deletions in the eukaryotic genome. The combination of popularity and superior capabilities of CRISPR/Cas9 system raises the possibility that this technology may occupy the roles currently served by RNAi and may even make RNAi obsolete. We performed a comparative analysis of the technical aspects and applications of the CRISPR/Cas9 system and RNAi in mammalian systems, with the purpose of charting out a predictive picture on whether the CRISPR/Cas9 system will eclipse the existence and future of RNAi. The conclusion drawn from this analysis is that RNAi will still occupy specific domains of biomedical research and clinical applications, under the current state of development of these technologies. However, further improvements in CRISPR/Cas9 based technology may ultimately enable it to dominate RNAi in the long term.

Heritable CRISPR/Cas9-mediated genome editing in the yellow fever mosquito, Aedes aegypti.

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Shengzhang Dong

Full Text Available In vivo targeted gene disruption is a powerful tool to study gene function. Thus far, two tools for genome editing in Aedes aegypti have been applied, zinc-finger nucleases (ZFN and transcription activator-like effector nucleases (TALEN. As a promising alternative to ZFN and TALEN, which are difficult to produce and validate using standard molecular biological techniques, the clustered regularly interspaced short palindromic repeats/CRISPR-associated sequence 9 (CRISPR/Cas9 system has recently been discovered as a "do-it-yourself" genome editing tool. Here, we describe the use of CRISPR/Cas9 in the mosquito vector, Aedes aegypti. In a transgenic mosquito line expressing both Dsred and enhanced cyan fluorescent protein (ECFP from the eye tissue-specific 3xP3 promoter in separated but tightly linked expression cassettes, we targeted the ECFP nucleotide sequence for disruption. When supplying the Cas9 enzyme and two sgRNAs targeting different regions of the ECFP gene as in vitro transcribed mRNAs for germline transformation, we recovered four different G1 pools (5.5% knockout efficiency where individuals still expressed DsRed but no longer ECFP. PCR amplification, cloning, and sequencing of PCR amplicons revealed indels in the ECFP target gene ranging from 2-27 nucleotides. These results show for the first time that CRISPR/Cas9 mediated gene editing is achievable in Ae. aegypti, paving the way for further functional genomics related studies in this mosquito species.

CRISPR Distribution within the Escherichia coli Species Is Not Suggestive of Immunity-Associated Diversifying Selection ▿ †

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Touchon, Marie; Charpentier, Sophie; Clermont, Olivier; Rocha, Eduardo P. C.; Denamur, Erick; Branger, Catherine

2011-01-01

In order to get further insights into the role of the clustered, regularly interspaced, short palindromic repeats (CRISPRs) in Escherichia coli, we analyzed the CRISPR diversity in a collection of 290 strains, in the phylogenetic framework of the strains represented by multilocus sequence typing (MLST). The set included 263 natural E. coli isolates exposed to various environments and isolated over a 20-year period from humans and animals, as well as 27 fully sequenced strains. Our analyses confirm that there are two largely independent pairs of CRISPR loci (CRISPR1 and -2 and CRISPR3 and -4), each associated with a different type of cas genes (Ecoli and Ypest, respectively), but that each pair of CRISPRs has similar dynamics. Strikingly, the major phylogenetic group B2 is almost devoid of CRISPRs. The majority of genomes analyzed lack Ypest cas genes and contain CRISPR3 with spacers matching Ypest cas genes. The analysis of relatedness between strains in terms of spacer repertoire and the MLST tree shows a pattern where closely related strains (MLST phylogenetic distance of 0.01) exhibit completely different CRISPRs. This suggests rare but radical turnover of spacers in CRISPRs rather than CRISPR gradual change. We found no link between the presence, size, or content of CRISPRs and the lifestyle of the strains. Our data suggest that, within the E. coli species, CRISPRs do not have the expected characteristics of a classical immune system. PMID:21421763

CRISPR distribution within the Escherichia coli species is not suggestive of immunity-associated diversifying selection.

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Touchon, Marie; Charpentier, Sophie; Clermont, Olivier; Rocha, Eduardo P C; Denamur, Erick; Branger, Catherine

2011-05-01

In order to get further insights into the role of the clustered, regularly interspaced, short palindromic repeats (CRISPRs) in Escherichia coli, we analyzed the CRISPR diversity in a collection of 290 strains, in the phylogenetic framework of the strains represented by multilocus sequence typing (MLST). The set included 263 natural E. coli isolates exposed to various environments and isolated over a 20-year period from humans and animals, as well as 27 fully sequenced strains. Our analyses confirm that there are two largely independent pairs of CRISPR loci (CRISPR1 and -2 and CRISPR3 and -4), each associated with a different type of cas genes (Ecoli and Ypest, respectively), but that each pair of CRISPRs has similar dynamics. Strikingly, the major phylogenetic group B2 is almost devoid of CRISPRs. The majority of genomes analyzed lack Ypest cas genes and contain CRISPR3 with spacers matching Ypest cas genes. The analysis of relatedness between strains in terms of spacer repertoire and the MLST tree shows a pattern where closely related strains (MLST phylogenetic distance of 0.01) exhibit completely different CRISPRs. This suggests rare but radical turnover of spacers in CRISPRs rather than CRISPR gradual change. We found no link between the presence, size, or content of CRISPRs and the lifestyle of the strains. Our data suggest that, within the E. coli species, CRISPRs do not have the expected characteristics of a classical immune system.

Polymorphism of CRISPR shows separated natural groupings of Shigella subtypes and evidence of horizontal transfer of CRISPR

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Yang, Chaojie; Li, Peng; Su, Wenli; Li, Hao; Liu, Hongbo; Yang, Guang; Xie, Jing; Yi, Shengjie; Wang, Jian; Cui, Xianyan; Wu, Zhihao; Wang, Ligui; Hao, Rongzhang; Jia, Leili; Qiu, Shaofu; Song, Hongbin

2015-01-01

Clustered, regularly interspaced, short palindromic repeats (CRISPR) act as an adaptive RNA-mediated immune mechanism in bacteria. They can also be used for identification and evolutionary studies based on polymorphisms within the CRISPR locus. We amplified and analyzed 6 CRISPR loci from 237 Shigella strains belonging to the 4 species groups, as well as 13 Escherichia coli strains. The CRISPR-associated (cas) gene sequence arrays of these strains were screened and compared. The CRISPR sequences from Shigella were conserved among subtypes, suggesting that CRISPR may represent a new identification tool for the detection and discrimination of Shigella species. Secondary structure analysis showed a different stem-loop structure at the terminal repeat, suggesting a distinct recognition mechanism in the formation of crRNA. In addition, the presence of “self-target” spacers and polymorphisms within CRISPR in Shigella indicated a selective pressure for inhibition of this system, which has the potential to damage “self DNA.” Homology analysis of spacers showed that CRISPR might be involved in the regulation of virulence transmission. Phylogenetic analysis based on CRISPR sequences from Shigella and E. coli indicated that although phenotypic properties maintain convergent evolution, the 4 Shigella species do not represent natural groupings. Surprisingly, comparative analysis of Shigella repeats with other species provided new evidence for CRISPR horizontal transfer. Our results suggested that CRISPR analysis is applicable for the detection of Shigella species and for investigation of evolutionary relationships. PMID:26327282

Engineered CRISPR Systems for Next Generation Gene Therapies.

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Pineda, Michael; Moghadam, Farzaneh; Ebrahimkhani, Mo R; Kiani, Samira

2017-09-15

An ideal in vivo gene therapy platform provides safe, reprogrammable, and precise strategies which modulate cell and tissue gene regulatory networks with a high temporal and spatial resolution. Clustered regularly interspaced short palindromic repeats (CRISPR), a bacterial adoptive immune system, and its CRISPR-associated protein 9 (Cas9), have gained attention for the ability to target and modify DNA sequences on demand with unprecedented flexibility and precision. The precision and programmability of Cas9 is derived from its complexation with a guide-RNA (gRNA) that is complementary to a desired genomic sequence. CRISPR systems open-up widespread applications including genetic disease modeling, functional screens, and synthetic gene regulation. The plausibility of in vivo genetic engineering using CRISPR has garnered significant traction as a next generation in vivo therapeutic. However, there are hurdles that need to be addressed before CRISPR-based strategies are fully implemented. Some key issues center on the controllability of the CRISPR platform, including minimizing genomic-off target effects and maximizing in vivo gene editing efficiency, in vivo cellular delivery, and spatial-temporal regulation. The modifiable components of CRISPR systems: Cas9 protein, gRNA, delivery platform, and the form of CRISPR system delivered (DNA, RNA, or ribonucleoprotein) have recently been engineered independently to design a better genome engineering toolbox. This review focuses on evaluating CRISPR potential as a next generation in vivo gene therapy platform and discusses bioengineering advancements that can address challenges associated with clinical translation of this emerging technology.

CRISPR/Cas9-mediated knock-in of an optimized TetO repeat for live cell imaging of endogenous loci.

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Tasan, Ipek; Sustackova, Gabriela; Zhang, Liguo; Kim, Jiah; Sivaguru, Mayandi; HamediRad, Mohammad; Wang, Yuchuan; Genova, Justin; Ma, Jian; Belmont, Andrew S; Zhao, Huimin

2018-06-15

Nuclear organization has an important role in determining genome function; however, it is not clear how spatiotemporal organization of the genome relates to functionality. To elucidate this relationship, a method for tracking any locus of interest is desirable. Recently clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) or transcription activator-like effectors were adapted for imaging endogenous loci; however, they are mostly limited to visualization of repetitive regions. Here, we report an efficient and scalable method named SHACKTeR (Short Homology and CRISPR/Cas9-mediated Knock-in of a TetO Repeat) for live cell imaging of specific chromosomal regions without the need for a pre-existing repetitive sequence. SHACKTeR requires only two modifications to the genome: CRISPR/Cas9-mediated knock-in of an optimized TetO repeat and its visualization by TetR-EGFP expression. Our simplified knock-in protocol, utilizing short homology arms integrated by polymerase chain reaction, was successful at labeling 10 different loci in HCT116 cells. We also showed the feasibility of knock-in into lamina-associated, heterochromatin regions, demonstrating that these regions prefer non-homologous end joining for knock-in. Using SHACKTeR, we were able to observe DNA replication at a specific locus by long-term live cell imaging. We anticipate the general applicability and scalability of our method will enhance causative analyses between gene function and compartmentalization in a high-throughput manner.

Metabolic Engineering of Probiotic Saccharomyces boulardii.

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Liu, Jing-Jing; Kong, In Iok; Zhang, Guo-Chang; Jayakody, Lahiru N; Kim, Heejin; Xia, Peng-Fei; Kwak, Suryang; Sung, Bong Hyun; Sohn, Jung-Hoon; Walukiewicz, Hanna E; Rao, Christopher V; Jin, Yong-Su

2016-04-01

Saccharomyces boulardiiis a probiotic yeast that has been used for promoting gut health as well as preventing diarrheal diseases. This yeast not only exhibits beneficial phenotypes for gut health but also can stay longer in the gut than Saccharomyces cerevisiae Therefore, S. boulardiiis an attractive host for metabolic engineering to produce biomolecules of interest in the gut. However, the lack of auxotrophic strains with defined genetic backgrounds has hampered the use of this strain for metabolic engineering. Here, we report the development of well-defined auxotrophic mutants (leu2,ura3,his3, and trp1) through clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-based genome editing. The resulting auxotrophic mutants can be used as a host for introducing various genetic perturbations, such as overexpression or deletion of a target gene, using existing genetic tools forS. cerevisiae We demonstrated the overexpression of a heterologous gene (lacZ), the correct localization of a target protein (red fluorescent protein) into mitochondria by using a protein localization signal, and the introduction of a heterologous metabolic pathway (xylose-assimilating pathway) in the genome ofS. boulardii We further demonstrated that human lysozyme, which is beneficial for human gut health, could be secreted by S. boulardii Our results suggest that more sophisticated genetic perturbations to improveS. boulardii can be performed without using a drug resistance marker, which is a prerequisite for in vivo applications using engineeredS. boulardii. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Detection and analysis of CRISPRs of Shigella.

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Guo, Xiangjiao; Wang, Yingfang; Duan, Guangcai; Xue, Zerun; Wang, Linlin; Wang, Pengfei; Qiu, Shaofu; Xi, Yuanlin; Yang, Haiyan

2015-01-01

The recently discovered CRISPRs (Clustered regularly interspaced short palindromic repeats) and Cas (CRISPR-associated) proteins are a novel genetic barrier that limits horizontal gene transfer in prokaryotes and the CRISPR loci provide a historical view of the exposure of prokaryotes to a variety of foreign genetic elements. The aim of study was to investigate the occurrence and distribution of the CRISPRs in Shigella. A collection of 61 strains of Shigella were screened for the existence of CRISPRs. Three CRISPR loci were identified among 61 shigella strains. CRISPR1/cas loci are detected in 49 strains of shigella. Yet, IS elements were detected in cas gene in some strains. In the remaining 12 Shigella flexneri strains, the CRISPR1/cas locus is deleted and only a cas3' pseudo gene and a repeat sequence are present. The presence of CRISPR2 is frequently accompanied by the emergence of CRISPR1. CRISPR3 loci were present in almost all strains (52/61). The length of CRISPR arrays varied from 1 to 9 spacers. Sequence analysis of the CRISPR arrays revealed that few spacers had matches in the GenBank databases. However, one spacer in CRISPR3 loci matches the cognate cas3 genes and no cas gene was present around CRISPR3 region. Analysis of CRISPR sequences show that CRISPR have little change which makes CRISPR poor genotyping markers. The present study is the first attempt to determine and analyze CRISPRs of shigella isolated from clinical patients.

Polymorphism of CRISPR shows separated natural groupings of Shigella subtypes and evidence of horizontal transfer of CRISPR.

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Yang, Chaojie; Li, Peng; Su, Wenli; Li, Hao; Liu, Hongbo; Yang, Guang; Xie, Jing; Yi, Shengjie; Wang, Jian; Cui, Xianyan; Wu, Zhihao; Wang, Ligui; Hao, Rongzhang; Jia, Leili; Qiu, Shaofu; Song, Hongbin

2015-01-01

Clustered, regularly interspaced, short palindromic repeats (CRISPR) act as an adaptive RNA-mediated immune mechanism in bacteria. They can also be used for identification and evolutionary studies based on polymorphisms within the CRISPR locus. We amplified and analyzed 6 CRISPR loci from 237 Shigella strains belonging to the 4 species groups, as well as 13 Escherichia coli strains. The CRISPR-associated (cas) gene sequence arrays of these strains were screened and compared. The CRISPR sequences from Shigella were conserved among subtypes, suggesting that CRISPR may represent a new identification tool for the detection and discrimination of Shigella species. Secondary structure analysis showed a different stem-loop structure at the terminal repeat, suggesting a distinct recognition mechanism in the formation of crRNA. In addition, the presence of "self-target" spacers and polymorphisms within CRISPR in Shigella indicated a selective pressure for inhibition of this system, which has the potential to damage "self DNA." Homology analysis of spacers showed that CRISPR might be involved in the regulation of virulence transmission. Phylogenetic analysis based on CRISPR sequences from Shigella and E. coli indicated that although phenotypic properties maintain convergent evolution, the 4 Shigella species do not represent natural groupings. Surprisingly, comparative analysis of Shigella repeats with other species provided new evidence for CRISPR horizontal transfer. Our results suggested that CRISPR analysis is applicable for the detection of Shigella species and for investigation of evolutionary relationships.

Suppression or knockout of SaF/SaM overcomes the Sa-mediated hybrid male sterility in rice.

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Xie, Yongyao; Niu, Baixiao; Long, Yunming; Li, Gousi; Tang, Jintao; Zhang, Yaling; Ren, Ding; Liu, Yao-Guang; Chen, Letian

2017-09-01

Hybrids between the indica and japonica subspecies of rice (Oryza sativa) are usually sterile, which hinders utilization of heterosis in the inter-subspecific hybrid breeding. The complex locus Sa comprises two adjacently located genes, SaF and SaM, which interact to cause abortion of pollen grains carrying the japonica allele in japonica-indica hybrids. Here we showed that silencing of SaF or SaM by RNA interference restored male fertility in indica-japonica hybrids with heterozygous Sa. We further used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing to knockout the SaF and SaM alleles, respectively, of an indica rice line to create hybrid-compatible lines. The resultant artificial neutral alleles did not affect pollen viability and other agricultural traits, but did break down the reproductive barrier in the hybrids. We found that some rice lines have natural neutral allele Sa-n, which was compatible with the typical japonica or indica Sa alleles in hybrids. Our results demonstrate that SaF and SaM are required for hybrid male sterility, but are not essential for pollen development. This study provides effective approaches for the generation of hybrid-compatible lines by knocking out the Sa locus or using the natural Sa-n allele to overcome hybrid male sterility in rice breeding. © 2017 The Authors. Bioelectromagnetics published by Wiley Periodicals, Inc. © 2017 Institute of Botany, Chinese Academy of Sciences.

The geometry of continuum regularization

International Nuclear Information System (INIS)

Halpern, M.B.

1987-03-01

This lecture is primarily an introduction to coordinate-invariant regularization, a recent advance in the continuum regularization program. In this context, the program is seen as fundamentally geometric, with all regularization contained in regularized DeWitt superstructures on field deformations