WorldWideScience

Sample records for service cas number

  1. Tenth anniversary of CAS ONLINE service : What CAS services should be in the new era of chemical information

    Science.gov (United States)

    Kostakos, Charles N.

    Chemical Abstracts Service celebrated 10th anniversary of CAS online information service in 1990. A speech given on the occasion reviewed history of the CAS ONLINE, in relation to its most important benefits for scientists and engineers. The development of STN international, the network through which CAS ONLINE is accessible around the world, was also discussed in the speech. The CAS ONLINE now contains a wide variety of files relating to chemical field including CA file, Registry file. CA previews,. CASREACT, CIN. MARPAT, etc for supplying chemical information worldwide.

  2. Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting | Office of Cancer Genomics

    Science.gov (United States)

    The CRISPR/Cas9 system enables genome editing and somatic cell genetic screens in mammalian cells. We performed genome-scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation/survival and found a strong correlation between increased gene copy number and decreased cell viability after genome editing. Within regions of copy-number gain, CRISPR/Cas9 targeting of both expressed and unexpressed genes, as well as intergenic loci, led to significantly decreased cell proliferation through induction of a G2 cell-cycle arrest.

  3. Les TIC au service du microcrédit : le cas des correspondants ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    Les TIC au service du microcrédit : le cas des correspondants bancaires au Brésil. Bien qu'il ait été prouvé que les programmes de microfinance constituaient un outil efficace pour lutter contre la pauvreté dans les pays en développement, ils tardent à prendre leur envol en Amérique latine, en particulier au Brésil.

  4. Utilization of chemical abstracts service (CAS) data bases

    International Nuclear Information System (INIS)

    Ehrhardt, F.; Gesellschaft Deutscher Chemiker, Berlin

    1979-04-01

    A method is developed describing the economic utilization of the Chemical Abstracts Service data bases CA Condensates and Chemical Abstracts Subject Index Alertin order to supplement the data base of the IDC-Inorganica-Documentationsystem built up to meet the peculiarities of the inorganic chemistry. The method consists of EDP-Programs and processes at which special authority files for coded compound and subject entries play an important role. One of the advantages of the method is that the intellectual effort necessary to create such a data base is reduced to a minimum. The authority files may be also used for orther purposes. (orig.) 891 WB 892 MB [de

  5. CRISPR/Cas9-mediated gene knockout is insensitive to target copy number but is dependent on guide RNA potency and Cas9/sgRNA threshold expression level.

    Science.gov (United States)

    Yuen, Garmen; Khan, Fehad J; Gao, Shaojian; Stommel, Jayne M; Batchelor, Eric; Wu, Xiaolin; Luo, Ji

    2017-11-16

    CRISPR/Cas9 is a powerful gene editing tool for gene knockout studies and functional genomic screens. Successful implementation of CRISPR often requires Cas9 to elicit efficient target knockout in a population of cells. In this study, we investigated the role of several key factors, including variation in target copy number, inherent potency of sgRNA guides, and expression level of Cas9 and sgRNA, in determining CRISPR knockout efficiency. Using isogenic, clonal cell lines with variable copy numbers of an EGFP transgene, we discovered that CRISPR knockout is relatively insensitive to target copy number, but is highly dependent on the potency of the sgRNA guide sequence. Kinetic analysis revealed that most target mutation occurs between 5 and 10 days following Cas9/sgRNA transduction, while sgRNAs with different potencies differ by their knockout time course and by their terminal-phase knockout efficiency. We showed that prolonged, low level expression of Cas9 and sgRNA often fails to elicit target mutation, particularly if the potency of the sgRNA is also low. Our findings provide new insights into the behavior of CRISPR/Cas9 in mammalian cells that could be used for future improvement of this platform. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

  6. Dana-Farber Cancer Institute (DFCI): Computational Correction of Copy-number Effect in CRISPR-Cas9 Essentiality Screens of Cancer Cells | Office of Cancer Genomics

    Science.gov (United States)

    Genome-wide CRISPR-Cas9 screens were performed in 341 cell lines. The results were processed with the CERES algorithm to produce copy-number and guide-efficacy corrected gene knockout effect estimates.

  7. RIFM fragrance ingredient safety assessment, isobornyl isovalerate, CAS registry number 7779-73-9.

    Science.gov (United States)

    Api, A M; Belsito, D; Bhatia, S; Bruze, M; Calow, P; Dagli, M L; Dekant, W; Fryer, A D; Kromidas, L; La Cava, S; Lapczynski, A; Liebler, D C; O'Brien, D; Parakhia, R; Penning, T M; Politano, V T; Ritacco, G; Salvito, D; Schultz, T W; Shen, J; Sipes, I G; Wall, B; Wilcox, D K

    2017-12-01

    This material was evaluated for genotoxicity, repeated dose toxicity, developmental toxicity, reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization potential, as well as, environmental safety. Data from the suitable read across analog isobornyl acetate (CAS # 125-12-2) show that this material is not genotoxic, provided a MOE > 100 for the repeated dose, developmental and reproductive endpoints, and does not have skin sensitization potential. The local respiratory toxicity endpoint was completed using the TTC (threshold of Toxicological Concern) for a Cramer Class II material (0.47 mg/day). The phototoxicity/photoallergenicity endpoint was completed based on suitable UV spectra. The environmental endpoint was completed as described in the RIFM Framework. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Computational correction of copy number effect improves specificity of CRISPR-Cas9 essentiality screens in cancer cells. | Office of Cancer Genomics

    Science.gov (United States)

    The CRISPR-Cas9 system has revolutionized gene editing both at single genes and in multiplexed loss-of-function screens, thus enabling precise genome-scale identification of genes essential for proliferation and survival of cancer cells. However, previous studies have reported that a gene-independent antiproliferative effect of Cas9-mediated DNA cleavage confounds such measurement of genetic dependency, thereby leading to false-positive results in copy number-amplified regions.

  9. 41 CFR 102-33.95 - What is the process for budgeting to acquire commercial aviation services (CAS)?

    Science.gov (United States)

    2010-07-01

    ... 41 Public Contracts and Property Management 3 2010-07-01 2010-07-01 false What is the process for budgeting to acquire commercial aviation services (CAS)? 102-33.95 Section 102-33.95 Public Contracts and... Parts The Process for Budgeting to Acquire Government Aircraft § 102-33.95 What is the process for...

  10. Service Provider Revenue Dependence of Offered Number of Service Classes

    Directory of Open Access Journals (Sweden)

    V. S. Aćimović-Raspopović

    2011-06-01

    Full Text Available In this paper possible applications of responsive pricing scheme and Stackelberg game for pricing telecommunication services with service provider as a leader and users acting as followers are analyzed. We have classified users according to an elasticity criterion into inelastic, partially elastic and elastic users. Their preferences are modelled through utility functions, which describe users’ sensitivity to changes in the quality of service and price. In the proposed algorithm a bandwidth management server is responsible for performing automatic optimal bandwidth allocation to each user’s session while maximizing its expected utility and the overall service provider’s revenue. The pricing algorithm is used for congestion control and more efficient network capacity utilization. We have analyzed different scenarios of the proposed usage-based pricing algorithm. Particularly, the influence of the number of service classes on price setting in terms of service provider’s revenue and total users’ utility maximization are discussed. The model is verified through numerous simulations performed by software that we have developed for that purpose.

  11. Social Security Number Verification Service (SSNVS)

    Data.gov (United States)

    Social Security Administration — SSNVS is a service offered by SSA's Business Services Online (BSO). It is used by employers and certain third-party submitters to verify the accuracy of the names...

  12. 76 FR 72124 - Internet-Based Telecommunications Relay Service Numbering

    Science.gov (United States)

    2011-11-22

    ... Docket No. 10-191; FCC 11-123] Internet-Based Telecommunications Relay Service Numbering AGENCY: Federal..., the information collection associated with the Commission's Internet- Based Telecommunications Relay... Telecommunications Relay Service Numbering, CG Docket No. 03-123; WC Docket No. 05-196; WC Docket No. 10-191; FCC 11...

  13. Perspectives d'introduction d'un marketing des services au sein des banques publiques Algériennes : Cas de la B.A.D.R.

    OpenAIRE

    Lachachi-taleb, Meriem

    2014-01-01

    Le premier chapitre présente les fondements théoriques relatifs au marketing des services ainsi qu'au concept de servuction et enfin au marketing mix des services dans le deuxiéme chapitre.Le troisiéme chapitre est consacré au marketing bancaire et au plan marketing dans le chapitre suivant, une étude de cas au sein de la B.A.D.R.Banque.

  14. 77 FR 1039 - Internet-Based Telecommunications Relay Service Numbering

    Science.gov (United States)

    2012-01-09

    ... FEDERAL COMMUNICATIONS COMMISSION 47 CFR Part 64 [WC Docket No. 10-191; Report No. 2939] Internet... toll-free numbers by users of Internet- based Telecommunications Relay Services (iTRS). DATES... any rules of particular applicability. Subject: Internet-Based Telecommunications Relay Service...

  15. 78 FR 36725 - Numbering Policies for Modern Communications; IP-Enabled Services; Telephone Number Requirements...

    Science.gov (United States)

    2013-06-19

    ... help speed the delivery of innovative services to consumers and businesses, while preserving the... available for public inspection during regular business hours in the FCC Reference Information Center... already broken the historical tie between a number and a specific device. For example, Skype permits users...

  16. Cas Wepener

    African Journals Online (AJOL)

    Owner

    Dubbelfoto is die eerste kortverhaalbundel van die teoloog Cas Wepener wat tot dusver veral akademiese artikels en godsdienstige boeke geskryf het. Die bundel se titel gee besondere prominensie aan die gegewe van die foto ter- wyl die motto wat gehaal is uit Roland Barthes se Camera Lucida die aandag vestig op die ...

  17. The CAS Classroom

    Science.gov (United States)

    Garner, Sue

    2004-01-01

    The Victorian Curriculum and Assessment Authority (VCAA) Computer Algebra System (CAS)Pilot study (2001-2005) is monitoring the use of CAS in senior secondary mathematics. This article explores the author's experiences in the CAS classroom and delineates changes in teaching style, as a result of the introduction of CAS into the senior mathematics…

  18. [CRISPR/CAS9, the King of Genome Editing Tools].

    Science.gov (United States)

    Bannikov, A V; Lavrov, A V

    2017-01-01

    The discovery of CRISPR/Cas9 brought a hope for having an efficient, reliable, and readily available tool for genome editing. CRISPR/Cas9 is certainly easy to use, while its efficiency and reliability remain the focus of studies. The review describes the general principles of the organization and function of Cas nucleases and a number of important issues to be considered while planning genome editing experiments with CRISPR/Cas9. The issues include evaluation of the efficiency and specificity for Cas9, sgRNA selection, Cas9 variants designed artificially, and use of homologous recombination and nonhomologous end joining in DNA editing.

  19. Real numbers tell real stories in health services management.

    Science.gov (United States)

    Heenan, Michael; Wood, Brady; Taylor, D Wayne

    2010-01-01

    In the words of one hospital manager, "hospital data is currently indigestible and alien to the average user." Drawing upon the experience of an academic hospital that, contrary to established practice, published real numbers alongside rates and ratios during a Clostridium difficile outbreak, the authors examined the pitfalls of publishing only abstract performance measures and the advantages of releasing real numbers to the public. This article identifies lessons for hospital board governance, media relations, employee communications, and citizen and patient engagement that are applicable across the healthcare industry in many countries. If healthcare is to be a caring industry, then care should be taken in the public reporting of data and information.

  20. RIFM fragrance ingredient safety assessment, acetic acid, C7-9-branched alkyl esters, C8-rich, CAS Registry Number 108419-32-5.

    Science.gov (United States)

    Api, A M; Belsito, D; Botelho, D; Browne, D; Bruze, M; Burton, G A; Buschmann, J; Calow, P; Dagli, M L; Date, M; Dekant, W; Deodhar, C; Fryer, A D; Joshi, K; La Cava, S; Lapczynski, A; Liebler, D C; O'Brien, D; Parakhia, R; Patel, A; Penning, T M; Ritacco, G; Romine, J; Salvito, D; Schultz, T W; Sipes, I G; Thakkar, Y; Tsang, S; Wahler, J

    2017-12-01

    The use of this material under current conditions is supported by existing information. This material was evaluated for genotoxicity, repeated dose toxicity, developmental and reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, as well as environmental safety. Data show that this material is not genotoxic. Data from the suitable read across analog isoamyl acetate (CAS# 123-92-2) show that this material does not have skin sensitization potential. The reproductive and local respiratory toxicity endpoints were completed using the TTC (Threshold of Toxicological Concern) for a Cramer Class I material (0.03 mg/kg/day and 1.4 mg/day, respectively). The repeated dose and developmental endpoint was completed using data on the target material, which provided a MOE > 100. The phototoxicity/photoallergenicity endpoint was completed based on suitable UV spectra. The environmental endpoint was completed as described in the RIFM Framework. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Situation a-didactique et dispositif d’apprentissage instrumenté : cas de construction de projets de service

    Directory of Open Access Journals (Sweden)

    Bernard Coulibaly

    2011-01-01

    Full Text Available Dans cette contribution nous tentons d’analyser une situation didactique instrumentée sur une plate forme d’apprentissage à distance, situation dans laquelle des étudiants de Master 2 professionnel sont invités à réaliser un projet de service fictif. Cet apprentissage s’opère en groupe de 3 à 4 étudiants et vise l’acquisition de compétences en matière de conduite de projet de service dans le domaine de l’ingénierie en milieu socio-éducatif. L’article analyse, à partir des traces d’interaction, le processus de construction de ces savoir-faire en management de projet. Il met en évidence les stratégies de contournement et d’adaptation que les ��tudiants élaborent pour échapper aux contraintes d’une situation didactique fondée sur un dispositif pédagogique instrumenté .Il permet ainsi de comprendre le rôle que des apprenants peuvent jouer dans la conception d’une situation didactique.In this paper we analyze an instrumented didactic situation that takes place on a distance learning platform. In this situation students in a professional master’s degree curriculum are invited to create a fictitious service project. They have to achieve this task in groups of 3 to 4 students. The task aims at making students acquire competences in terms of project management in the field of educational engineering. The article analyzes the process of building these skills in project management from traces of interaction. It highlights the strategies of circumvention and adaptation that students develop to escape the constraints of a teaching situation based on an instrumented educational device. It helps understand the role that learners can play in the design of a teaching situation.

  2. Ponction biopsies rénales dans le Service de Néphrologie de Fès: indications et résultats: à propos de 522 cas

    Science.gov (United States)

    Mbarki, Houda; Belghiti, Khadija Alaoui; Harmouch, Taoufiq; Najdi, Adil; Arrayhani, Mohamed; Sqalli, Tarik

    2016-01-01

    L'apport de la ponction biopsie rénale (PBR) dans le diagnostic, le choix thérapeutique et l’évaluation pronostique des néphropathies est considérable. Aucune étude marocaine n'a évalué la pratique et l'apport de la PBR. Notre objectif est d’étudier les indications de la PBR, déterminer la fréquence des maladies rénales identifiées par PBR dans notre région et de faire une confrontation entre les données clinico-biologiques et le diagnostic historique. Notre étude menée entre Janvier 2009 et Décembre 2012, est rétrospective. Nous avons inclus tous les patients du service de Néphrologie du CHU Hassan II de Fès ayant bénéficié d'une biopsie de reins natifs. 522 PBR ont été réalisées. Nous avons exclu 8 biopsies devant le manque de renseignements et avons donc retenu 514. L’âge moyen des patients au moment de la PBR est de 39 ±17 ans (3-82 ans). Le sex ratio est de 0,9. Le syndrome néphrotique est le diagnostic clinique le plus fréquent à tous les âges (58,2%). Les néphropathies glomérulaires représentent 94,2% des maladies rénales diagnostiquées, leur distribution varie selon l’âge des patients. La PBR a confirmé le premier diagnostic suspecté cliniquement dans 40,65% des cas, alors qu'elle a révélé un diagnostic inattendu chez 22,5% d'entre eux. Le diagnostic syndromique permet d'orienter vers la maladie rénale la plus probable et de guider les thérapeutiques urgentes en attendant les résultats de la PBR. Mais il ne peut en aucun remplacer la PBR qui reste le gold standard. PMID:27583085

  3. Describing Pre-Service Teachers' Developing Understanding of Elementary Number Theory Topics

    Science.gov (United States)

    Feldman, Ziv

    2012-01-01

    Although elementary number theory topics are closely linked to foundational topics in number and operations and are prevalent in elementary and middle grades mathematics curricula, little is currently known about how students and teachers make sense of them. This study investigated pre-service elementary teachers' developing understanding of…

  4. Strategies of Pre-Service Primary School Teachers for Solving Addition Problems with Negative Numbers

    Science.gov (United States)

    Almeida, Rut; Bruno, Alicia

    2014-01-01

    This paper analyses the strategies used by pre-service primary school teachers for solving simple addition problems involving negative numbers. The findings reveal six different strategies that depend on the difficulty of the problem and, in particular, on the unknown quantity. We note that students use negative numbers in those problems they find…

  5. Complementary Information Derived from CRISPR Cas9 Mediated Gene Deletion and Suppression. | Office of Cancer Genomics

    Science.gov (United States)

    CRISPR-Cas9 provides the means to perform genome editing and facilitates loss-of-function screens. However, we and others demonstrated that expression of the Cas9 endonuclease induces a gene-independent response that correlates with the number of target sequences in the genome. An alternative approach to suppressing gene expression is to block transcription using a catalytically inactive Cas9 (dCas9). Here we directly compare genome editing by CRISPR-Cas9 (cutting, CRISPRc) and gene suppression using KRAB-dCas9 (CRISPRi) in loss-of-function screens to identify cell essential genes.

  6. On the Integration of Computer Algebra Systems (CAS) by Canadian Mathematicians: Results of a National Survey

    Science.gov (United States)

    Buteau, Chantal; Jarvis, Daniel H.; Lavicza, Zsolt

    2014-01-01

    In this article, we outline the findings of a Canadian survey study (N = 302) that focused on the extent of computer algebra systems (CAS)-based technology use in postsecondary mathematics instruction. Results suggest that a considerable number of Canadian mathematicians use CAS in research and teaching. CAS use in research was found to be the…

  7. Asteroid named after CAS scientist

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    @@ An asteroid has been named after CAS astronomy historian XI Zezong with the approval of the International Minor Planet Nomenclature Committee (IMPNC), announced China's National Astronomical Observatories at CAS (NAOC) on 17 August.

  8. CRISPR-Cas

    NARCIS (Netherlands)

    Jackson, Simon A.; McKenzie, Rebecca E.; Fagerlund, Robert D.; Kieper, Sebastian N.; Fineran, Peter C.; Brouns, Stan J.J.

    2017-01-01

    Bacteria and archaea are engaged in a constant arms race to defend against the ever-present threats of viruses and invasion by mobile genetic elements. The most flexible weapons in the prokaryotic defense arsenal are the CRISPR-Cas adaptive immune systems. These systems are capable of selective

  9. Not all predicted CRISPR-Cas systems are equal: isolated cas genes and classes of CRISPR like elements.

    Science.gov (United States)

    Zhang, Quan; Ye, Yuzhen

    2017-02-06

    The CRISPR-Cas systems in prokaryotes are RNA-guided immune systems that target and deactivate foreign nucleic acids. A typical CRISPR-Cas system consists of a CRISPR array of repeat and spacer units, and a locus of cas genes. The CRISPR and the cas locus are often located next to each other in the genomes. However, there is no quantitative estimate of the co-location. In addition, ad-hoc studies have shown that some non-CRISPR genomic elements contain repeat-spacer-like structures and are mistaken as CRISPRs. Using available genome sequences, we observed that a significant number of genomes have isolated cas loci and/or CRISPRs. We found that 11%, 22% and 28% of the type I, II and III cas loci are isolated (without CRISPRs in the same genomes at all or with CRISPRs distant in the genomes), respectively. We identified a large number of genomic elements that superficially reassemble CRISPRs but don't contain diverse spacers and have no companion cas genes. We called these elements false-CRISPRs and further classified them into groups, including tandem repeats and Staphylococcus aureus repeat (STAR)-like elements. This is the first systematic study to collect and characterize false-CRISPR elements. We demonstrated that false-CRISPRs could be used to reduce the false annotation of CRISPRs, therefore showing them to be useful for improving the annotation of CRISPR-Cas systems.

  10. Service hall in Number 1 Fukushima Nuclear Power Station, Tokyo Electric Power Company, Inc

    International Nuclear Information System (INIS)

    Tawara, Shigesuke

    1979-01-01

    There are six BWR type nuclear power plants in the Number 1 Fukushima Nuclear Power Station, Tokyo Electric Power Company, Inc. The service hall of the station is located near the entrance of the station. In the center of this service hall, there is the model of a nuclear reactor of full scale. This mock-up shows the core region in the reactor pressure vessel for the number one plant. The diameter and the thickness of the pressure vessel are about 5 m and 16 cm, respectively. The fuel assemblies and control rods are set just like the actual reactor, and the start-up operation of the reactor is shown colorfully and dynamically by pushing a button. When the control rods are pulled out, the boiling of water is demonstrated. The 1/50 scale model of the sixth plant with the power generating capacity of 1100 MWe is set, and this model is linked to the mock-up of reactor written above. The operations of a recirculating loop, a turbine and a condenser are shown by switching on and off lamps. The other exhibitions are shielding concrete wall, ECCS model, and many kinds of panels and models. This service hall is incorporated in the course of study and observation of civics. The good environmental effects to fishes and shells are explained in this service hall. Official buildings and schools are built near the service hall utilizing the tax and grant concerning power generation. This service hall contributes to give much freedom from anxiety to the public by the tour. (Nakai, Y.)

  11. Recent Advances in Genome Editing Using CRISPR/Cas9

    Science.gov (United States)

    Ding, Yuduan; Li, Hong; Chen, Ling-Ling; Xie, Kabin

    2016-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system is a versatile tool for genome engineering that uses a guide RNA (gRNA) to target Cas9 to a specific sequence. This simple RNA-guided genome-editing technology has become a revolutionary tool in biology and has many innovative applications in different fields. In this review, we briefly introduce the Cas9-mediated genome-editing method, summarize the recent advances in CRISPR/Cas9 technology, and discuss their implications for plant research. To date, targeted gene knockout using the Cas9/gRNA system has been established in many plant species, and the targeting efficiency and capacity of Cas9 has been improved by optimizing its expression and that of its gRNA. The CRISPR/Cas9 system can also be used for sequence-specific mutagenesis/integration and transcriptional control of target genes. We also discuss off-target effects and the constraint that the protospacer-adjacent motif (PAM) puts on CRISPR/Cas9 genome engineering. To address these problems, a number of bioinformatic tools are available to help design specific gRNAs, and new Cas9 variants and orthologs with high fidelity and alternative PAM specificities have been engineered. Owing to these recent efforts, the CRISPR/Cas9 system is becoming a revolutionary and flexible tool for genome engineering. Adoption of the CRISPR/Cas9 technology in plant research would enable the investigation of plant biology at an unprecedented depth and create innovative applications in precise crop breeding. PMID:27252719

  12. New CRISPR-Cas systems from uncultivated microbes

    Science.gov (United States)

    Burstein, David; Harrington, Lucas B.; Strutt, Steven C.; Probst, Alexander J.; Anantharaman, Karthik; Thomas, Brian C.; Doudna, Jennifer A.; Banfield, Jillian F.

    2017-02-01

    CRISPR-Cas systems provide microbes with adaptive immunity by employing short DNA sequences, termed spacers, that guide Cas proteins to cleave foreign DNA. Class 2 CRISPR-Cas systems are streamlined versions, in which a single RNA-bound Cas protein recognizes and cleaves target sequences. The programmable nature of these minimal systems has enabled researchers to repurpose them into a versatile technology that is broadly revolutionizing biological and clinical research. However, current CRISPR-Cas technologies are based solely on systems from isolated bacteria, leaving the vast majority of enzymes from organisms that have not been cultured untapped. Metagenomics, the sequencing of DNA extracted directly from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms. Here, using genome-resolved metagenomics, we identify a number of CRISPR-Cas systems, including the first reported Cas9 in the archaeal domain of life, to our knowledge. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, we discovered two previously unknown systems, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. Notably, all required functional components were identified by metagenomics, enabling validation of robust in vivo RNA-guided DNA interference activity in Escherichia coli. Interrogation of environmental microbial communities combined with in vivo experiments allows us to access an unprecedented diversity of genomes, the content of which will expand the repertoire of microbe-based biotechnologies.

  13. Soil Conservation Service Curve Number method: How to mend a wrong soil moisture accounting procedure?

    Science.gov (United States)

    Michel, Claude; Andréassian, Vazken; Perrin, Charles

    2005-02-01

    This paper unveils major inconsistencies in the age-old and yet efficient Soil Conservation Service Curve Number (SCS-CN) procedure. Our findings are based on an analysis of the continuous soil moisture accounting procedure implied by the SCS-CN equation. It is shown that several flaws plague the original SCS-CN procedure, the most important one being a confusion between intrinsic parameter and initial condition. A change of parameterization and a more complete assessment of the initial condition lead to a renewed SCS-CN procedure, while keeping the acknowledged efficiency of the original method.

  14. Long-term hydrological simulation based on the Soil Conservation Service curve number

    Science.gov (United States)

    Mishra, Surendra Kumar; Singh, Vijay P.

    2004-05-01

    Presenting a critical review of daily flow simulation models based on the Soil Conservation Service curve number (SCS-CN), this paper introduces a more versatile model based on the modified SCS-CN method, which specializes into seven cases. The proposed model was applied to the Hemavati watershed (area = 600 km2) in India and was found to yield satisfactory results in both calibration and validation. The model conserved monthly and annual runoff volumes satisfactorily. A sensitivity analysis of the model parameters was performed, including the effect of variation in storm duration. Finally, to investigate the model components, all seven variants of the modified version were tested for their suitability.

  15. Empirical solution of Green-Ampt equation using soil conservation service - curve number values

    Science.gov (United States)

    Grimaldi, S.; Petroselli, A.; Romano, N.

    2012-09-01

    The Soil Conservation Service - Curve Number (SCS-CN) method is a popular widely used rainfall-runoff model for quantifying the total stream-flow volume generated by storm rainfall, but its application is not appropriate for sub-daily resolutions. In order to overcome this drawback, the Green-Ampt (GA) infiltration equation is considered and an empirical solution is proposed and evaluated. The procedure, named CN4GA (Curve Number for Green-Ampt), aims to calibrate the Green-Ampt model parameters distributing in time the global information provided by the SCS-CN method. The proposed procedure is evaluated by analysing observed rainfall-runoff events; results show that CN4GA seems to provide better agreement with the observed hydrographs respect to the classic SCS-CN method.

  16. How type II CRISPR-Cas establish immunity through Cas1-Cas2-mediated spacer integration.

    Science.gov (United States)

    Xiao, Yibei; Ng, Sherwin; Nam, Ki Hyun; Ke, Ailong

    2017-10-05

    CRISPR (clustered regularly interspaced short palindromic repeats) and the nearby Cas (CRISPR-associated) operon establish an RNA-based adaptive immunity system in prokaryotes. Molecular memory is created when a short foreign DNA-derived prespacer is integrated into the CRISPR array as a new spacer. Whereas the RNA-guided CRISPR interference mechanism varies widely among CRISPR-Cas systems, the spacer integration mechanism is essentially identical. The conserved Cas1 and Cas2 proteins form an integrase complex consisting of two distal Cas1 dimers bridged by a Cas2 dimer. The prespacer is bound by Cas1-Cas2 as a dual-forked DNA, and the terminal 3'-OH of each 3' overhang serves as an attacking nucleophile during integration. The prespacer is preferentially integrated into the leader-proximal region of the CRISPR array, guided by the leader sequence and a pair of inverted repeats inside the CRISPR repeat. Spacer integration in the well-studied Escherichia coli type I-E CRISPR system also relies on the bacterial integration host factor. In type II-A CRISPR, however, Cas1-Cas2 alone integrates spacers efficiently in vitro; other Cas proteins (such as Cas9 and Csn2) have accessory roles in the biogenesis phase of prespacers. Here we present four structural snapshots from the type II-A system of Enterococcus faecalis Cas1 and Cas2 during spacer integration. Enterococcus faecalis Cas1-Cas2 selectively binds to a splayed 30-base-pair prespacer bearing 4-nucleotide 3' overhangs. Three molecular events take place upon encountering a target: first, the Cas1-Cas2-prespacer complex searches for half-sites stochastically, then it preferentially interacts with the leader-side CRISPR repeat, and finally, it catalyses a nucleophilic attack that connects one strand of the leader-proximal repeat to the prespacer 3' overhang. Recognition of the spacer half-site requires DNA bending and leads to full integration. We derive a mechanistic framework to explain the stepwise spacer

  17. Payments for Environmental Services: Can They Work? Systèmes de paiement pour services environnementaux : une solution efficace ? Le cas du Mexique Pago por Servicios Ambientales: ¿Pueden funcionar? El caso de México

    Directory of Open Access Journals (Sweden)

    Helena García Romero

    2012-06-01

    Full Text Available Payments for Environmental Services (PES Programs can be useful policy instruments for achieving conservation objectives through incentive mechanisms.  However, the success of such programs depends on the particular solutions that are given to political economy constraints and challenges. The Mexican case provides helpful lessons on this topic, in addition to design and implementation insights.The Mexican PES program has been in place since 2003.  It strives to protect well-conserved forests and to have a social impact through payments made to the most marginalized communities.  Nevertheless, its impact in terms of avoided deforestation is not very high. This is due to targeting failures that arise from an internal trade-off between social and conservation goals.  In order to align both objectives the continuous negotiation of goals and targeting mechanisms between the different stakeholders: government, bureaucracy, non-government organizations (NGOs, and local communities must be kept in mind. Understanding and anticipating this policy process can ensure the desirable outcomes of PES programs in terms of poverty alleviation and conservation.Les programmes de systèmes de paiement pour services environnementaux (PES - Payments for Environmental Services peuvent être des instruments d’intervention efficaces pour atteindre les objectifs en matière de conservation des écosystèmes grâce aux mesures d’incitation qu’ils englobent. La réussite de ce type de programmes dépend toutefois des solutions spécifiques mises en place pour répondre aux contraintes et problématiques de l’économie politique. Le cas du Mexique permet de tirer des enseignements utiles à ce sujet et donne des éclairages sur la manière de concevoir et de mettre en oeuvre ces programmes.Le programme PES est mis en oeuvre au Mexique depuis 2003. Il vise à protéger les forêts préservées et à avoir un impact social en rémunérant les communautés les plus

  18. IMPLICATIONS ON THE NUMBER AND CONCENTRATION OF SUPPLIERS ON ROMANIAN TRANSPORT SERVICES

    Directory of Open Access Journals (Sweden)

    PĂUN IULIAN GABRIEL

    2017-02-01

    Full Text Available This paper aims to capture the current situation characterizing the transport sector in Romania by degree of concentration of transport service providers. Market analysis covers road, rail and air transport. Knowing the concentration of these markets is useful when it realizes development strategy of these sectors with profound implications on the national economy. There is a decreasing degree of concentration in the road transport sector due to growing number of enterprises in this market. Air transport has similar trend the road transport, the concentration declining during the span of years analyzed. Regarding the Herfindahl-Hirschmann index in the railway sector, we find that it reveals for the year 2012 a very high degree of concentration.

  19. Evaluation of the Soil Conservation Service curve number methodology using data from agricultural plots

    Science.gov (United States)

    Lal, Mohan; Mishra, S. K.; Pandey, Ashish; Pandey, R. P.; Meena, P. K.; Chaudhary, Anubhav; Jha, Ranjit Kumar; Shreevastava, Ajit Kumar; Kumar, Yogendra

    2017-01-01

    The Soil Conservation Service curve number (SCS-CN) method, also known as the Natural Resources Conservation Service curve number (NRCS-CN) method, is popular for computing the volume of direct surface runoff for a given rainfall event. The performance of the SCS-CN method, based on large rainfall (P) and runoff (Q) datasets of United States watersheds, is evaluated using a large dataset of natural storm events from 27 agricultural plots in India. On the whole, the CN estimates from the National Engineering Handbook (chapter 4) tables do not match those derived from the observed P and Q datasets. As a result, the runoff prediction using former CNs was poor for the data of 22 (out of 24) plots. However, the match was little better for higher CN values, consistent with the general notion that the existing SCS-CN method performs better for high rainfall-runoff (high CN) events. Infiltration capacity (fc) was the main explanatory variable for runoff (or CN) production in study plots as it exhibited the expected inverse relationship between CN and fc. The plot-data optimization yielded initial abstraction coefficient (λ) values from 0 to 0.659 for the ordered dataset and 0 to 0.208 for the natural dataset (with 0 as the most frequent value). Mean and median λ values were, respectively, 0.030 and 0 for the natural rainfall-runoff dataset and 0.108 and 0 for the ordered rainfall-runoff dataset. Runoff estimation was very sensitive to λ and it improved consistently as λ changed from 0.2 to 0.03.

  20. Mental Health Services Use Predicted by Number of Mental Health Problems and Gender in a Total Population Study

    Directory of Open Access Journals (Sweden)

    Maj-Britt Posserud

    2013-01-01

    Full Text Available We examined the relationship between service use and the number of problem areas as reported by parents and teachers on questionnaires among children aged 7–9 years old in the Bergen Child Study, a total population study including more than 9000 children. A problem area was counted as present if the child scored above the 95th percentile on parent and/or teacher questionnaire. A total number of 13 problem areas were included. Odd ratios (ORs for contact with child and adolescent mental health services (CAMH, school psychology services (SPS, health visiting nurse/physician, and school support were calculated with gender as covariate. The number of symptom areas was highly predictive of service use, showing a dose-response relationship for all services. Children scoring on ≥4 problem areas had a more than hundredfold risk of being in contact with CAMH services compared to children without problems. The mean number of problem areas for children in CAMH and SPS was 6.1 and 4.4 respectively, strongly supporting the ESSENCE model predicting multisymptomatology in children in specialized services. Even after controlling for number of problem areas, boys were twice as likely as girls to be in contact with CAMH, replicating previous findings of female gender being a strong barrier to mental health services.

  1. La Greffe de Peau dans le Traitement des Sequelles de la Main Brulee. A Propos de 152 Cas - Experience du Service de Chirurgie Plastique du Centre Hospitalier Universitaire Ibn-Sina, Rabat, Maroc

    Science.gov (United States)

    El Mazouz, S.; Fejjal, N.; Hafidi, J.; Cherkab, L.; Mejjati, H.; Belfqih, R.; Gharib, N.; Abbassi, A.

    2010-01-01

    Summary La main est fréquemment exposée aux brûlures, entraînant des séquelles esthétiques et fonctionnelles. Le traitement de ces séquelles est surtout chirurgical et consiste en la greffe de peau, dont le type dépend de la localisation de la brûlure et du type des séquelles. Dans ce travail rétrospectif, nous rapportons une série de 152 cas de brûlures des mains colligés au service de chirurgie plastique du Centre Hospitalier Universitaire Ibn-Sina de Rabat sur une période de dix ans, allant de 1998 à 2007. Les indications thérapeutiques dépendent du type de séquelles et de la localisation de la brûlure. En tout, 97 patients ont bénéficié d'une greffe cutanée, dont 76% par greffe de peau totale, 21% par greffe de peau demi-épaisse et 3% par peau fine. Les séquelles des brûlures des mains posent un problème thérapeutique majeur, malgré la diversité des procédés chirurgicaux, d'où l'intérêt de la prévention. PMID:21991196

  2. CAS School in Germany

    CERN Multimedia

    CERN Accelerator School

    The CERN Accelerator School (CAS), the Helmholtz Centre for Heavy Ion Research GmbH (GSI) and the Technische Universität Darmstadt (TU Darmstadt) jointly organised a course on General Accelerator Physics, at intermediate level, at TU Darmstadt from 27 September to 9 October 2009.   Participants in the CERN Accelerator School in Darmstadt, Germany. The Intermediate-level course followed established practice, with lectures on core topics in the mornings and specialised courses in the afternoons. The latter provided "hands-on" education and experience in the three selected topics: "RF Measurement Techniques", "Beam Instrumentation and Diagnostics" and "Optics Design and Correction". These proved to be highly successful, with participants choosing one course and following the topic throughout the school. Guided studies, tutorials, seminars and a poster session completed the programme. A visit to GSI and the F...

  3. Michigan Journal of Community Service Learning. Volume 13, Number 1, Fall 2006

    Science.gov (United States)

    Howard, Jeffrey, Ed.

    2006-01-01

    The "Michigan Journal of Community Service Learning" ("MJCSL") is a national, peer-reviewed journal consisting of articles written by faculty and service-learning educators on research, theory, pedagogy, and issues pertinent to the service-learning community. The "MJCSL" aims to: (1) widen the community of…

  4. Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition.

    Science.gov (United States)

    Kleinstiver, Benjamin P; Prew, Michelle S; Tsai, Shengdar Q; Nguyen, Nhu T; Topkar, Ved V; Zheng, Zongli; Joung, J Keith

    2015-12-01

    CRISPR-Cas9 nucleases target specific DNA sequences using a guide RNA but also require recognition of a protospacer adjacent motif (PAM) by the Cas9 protein. Although longer PAMs can potentially improve the specificity of genome editing, they limit the range of sequences that Cas9 orthologs can target. One potential strategy to relieve this restriction is to relax the PAM recognition specificity of Cas9. Here we used molecular evolution to modify the NNGRRT PAM of Staphylococcus aureus Cas9 (SaCas9). One variant we identified, referred to as KKH SaCas9, showed robust genome editing activities at endogenous human target sites with NNNRRT PAMs, thereby increasing SaCas9 targeting range by two- to fourfold. Using GUIDE-seq, we show that wild-type and KKH SaCas9 induce comparable numbers of off-target effects in human cells. Our strategy for evolving PAM specificity does not require structural information and therefore should be applicable to a wide range of Cas9 orthologs.

  5. DIESEL ENGINE SYSTEMS. AGRICULTURAL MACHINERY--SERVICE OCCUPATIONS, MODULE NUMBER 15.

    Science.gov (United States)

    Ohio State Univ., Columbus. Center for Vocational and Technical Education.

    ONE OF A SERIES DESIGNED TO HELP TEACHERS PREPARE POSTSECONDARY STUDENTS FOR AGRICULTURAL MACHINERY SERVICE OCCUPATIONS AS PARTS MEN, MECHANICS, MECHANIC'S HELPERS, AND SERVICE SUPERVISORS, THIS GUIDE AIMS TO DEVELOP STUDENT UNDERSTANDING OF THE CONSTRUCTION AND OPERATING PRINCIPLES OF DIESEL ENGINES. IT WAS DEVELOPED BY A NATIONAL TASK FORCE ON…

  6. 41 CFR 102-33.440 - Who must report CAS cost and utilization data?

    Science.gov (United States)

    2010-07-01

    ...-MANAGEMENT OF GOVERNMENT AIRCRAFT Reporting Information on Government Aircraft Commercial Aviation Services... agencies, except the Armed Forces and U.S. intelligence agencies, must report CAS cost and utilization data...

  7. The effect of relational constructs on customer referrals and number of services purchases from a multi-service provider: Does age of a relationship matter?

    NARCIS (Netherlands)

    Ph.H.B.F. Franses (Philip Hans); P.C. Verhoef (Peter); J.C. Hoekstra (Janny)

    2002-01-01

    textabstractThe authors examine the effect of relational constructs (e.g., satisfaction, trust, and affective and calculative commitment) on customer referrals and the number of services purchased, as well as the moderating effect of age of the relationship on these relationships. The research

  8. Corrélats des enquêtes conjointes des services de protection de l'enfance et des services de police sur les abus sexuels d'enfants : résultats de l'Étude canadienne sur l'incidence des signalements de cas de violence et de négligence envers les enfants 2008

    Directory of Open Access Journals (Sweden)

    L. Tonmyr

    2015-01-01

    Full Text Available Introduction : Dans cette étude, nous examinons la fréquence des enquêtes conjointes menées par les services de protection de l'enfance et les services de police dans les cas d'abus sexuels en comparaison des autres types de maltraitance. Nous examinons également les associations, dans les enquêtes conjointes, entre les caractéristiques relatives à l'enfant, celles relatives au pourvoyeur de soins de l'enfant, celles relatives aux mauvais traitements eux-mêmes et celles relatives à l'enquête, en nous intéressant plus particulièrement aux enquêtes sur les abus sexuels. Méthodologie : Nous avons analysé par régression logistique les données de l'Étude canadienne sur l'incidence des signalements de cas de violence et de négligence envers les enfants 2008. Résultats : D'après les données, les enquêtes conjointes portent en premier lieu sur les abus sexuels (55 %, puis sur la violence physique, la négligence et la violence psychologique. La corroboration des mauvais traitements, les mauvais traitements graves, le placement, l'intervention des tribunaux de la jeunesse et l'orientation d'un membre de la famille vers des services spécialisés sont plus fréquents quand les services de police participent à l'enquête. Conclusion : Cette étude vient bonifier l'information limitée dont on dispose sur les corrélats des enquêtes conjointes menées par les agences de protection de l'enfance et les services de police. D'autres recherches devront être effectuées pour déterminer l'efficacité de ces enquêtes conjointes.

  9. Pre-Service Teachers' Computational Knowledge, Efficacy, and Number Sense Skills

    Science.gov (United States)

    Hinton, Vanessa

    2011-01-01

    The National Council of Teachers of Mathematics (NCTM) Curriculum Focal Points (2006) suggested heavy emphasis on instruction in whole numbers for young elementary students. Any intervention curriculum for students who are at-risk for mathematic difficulties should not be oversimplified. Number sense was defined by Berch (1998) as a developing…

  10. 78 FR 56266 - Consent Based Social Security Number Verification (CBSV) Service

    Science.gov (United States)

    2013-09-12

    ... developed CBSV as a user- friendly, internet-based application with safeguards that protect the public's information. In addition to the benefit of providing high volume, centralized SSN verification services to users in a secure manner, CBSV provides us with cost and workload management benefits. New Information...

  11. 76 FR 60112 - Consent Based Social Security Number Verification (CBSV) Service

    Science.gov (United States)

    2011-09-28

    ... protect the public's information. In addition to the benefit of providing high volume, centralized SSN verification services to the business community in a secure manner, CBSV provides us with cost and workload management benefits. New Information: To use CBSV, interested parties must pay a one- time non-refundable...

  12. Service-Learning. National Dropout Prevention Center/Network Newsletter. Volume 22, Number 4

    Science.gov (United States)

    Duckenfield, Marty, Ed.

    2011-01-01

    The "National Dropout Prevention Newsletter" is published quarterly by the National Dropout Prevention Center/Network. This issue contains the following articles: (1) Dropouts and Democracy (Robert Shumer); (2) 2011 NDPN Crystal Star Winners; (3) Service-Learning as Dropout Intervention and More (Michael VanKeulen); and (4) Teacher…

  13. Tuning CRISPR-Cas9 Gene Drives in Saccharomyces cerevisiae

    Science.gov (United States)

    Roggenkamp, Emily; Giersch, Rachael M.; Schrock, Madison N.; Turnquist, Emily; Halloran, Megan; Finnigan, Gregory C.

    2018-01-01

    Control of biological populations is an ongoing challenge in many fields, including agriculture, biodiversity, ecological preservation, pest control, and the spread of disease. In some cases, such as insects that harbor human pathogens (e.g., malaria), elimination or reduction of a small number of species would have a dramatic impact across the globe. Given the recent discovery and development of the CRISPR-Cas9 gene editing technology, a unique arrangement of this system, a nuclease-based “gene drive,” allows for the super-Mendelian spread and forced propagation of a genetic element through a population. Recent studies have demonstrated the ability of a gene drive to rapidly spread within and nearly eliminate insect populations in a laboratory setting. While there are still ongoing technical challenges to design of a more optimal gene drive to be used in wild populations, there are still serious ecological and ethical concerns surrounding the nature of this powerful biological agent. Here, we use budding yeast as a safe and fully contained model system to explore mechanisms that might allow for programmed regulation of gene drive activity. We describe four conserved features of all CRISPR-based drives and demonstrate the ability of each drive component—Cas9 protein level, sgRNA identity, Cas9 nucleocytoplasmic shuttling, and novel Cas9-Cas9 tandem fusions—to modulate drive activity within a population. PMID:29348295

  14. L’usage politique de l’islam : l’universel au service d’un État. Le cas du Maroc The Political Use of Islam. The Universal in the Service of a State. The Moroccan Case

    Directory of Open Access Journals (Sweden)

    Abdessamad Belhaj

    2011-03-01

    Full Text Available Lorsqu’il s’agit d’étudier les rapports des pays arabes à la mondialisation, il est nécessaire d’évoquer l’islam. En tant que facteur local, la symbolique islamique offre des ressources de légitimation au pouvoir. Le cas marocain illustre bien la manière dont les rapports existant entre le Makhzen et le répertoire religieux (Amīr al-Mu’minīne, la bayca , la chérifibilité…  font de l’introduction de la modernité un simple espace virtuel et fonctionnel. L’émergence de la contestation islamiste participe à la consécration du répertoire traditionnel. D’autre part, depuis les trois dernières décennies, l’essor de l’islam mondialisé offre au Maroc des opportunités politiques à l’échelle mondiale. Ceci est observable au niveau du rôle joué par le pays dans le cadre de la diplomatie islamique (OCI, ISESCO…  ou au niveau de son positionnement dans l’islam européen. La gestion de l’interaction entre l’islam local et l’islam global, adaptée à la conjoncture internationale, assure le maintien au pouvoir du régime.One cannot study the relationships between Arab countries and globalization without taking Islam into account for it offers symbolic support to legitimizing local power. Morocco is a good example : the relations between the royal court (Makhzen and Islamic features such as being the Commander of the Faithful or Sheriffhood make of modernity a mere virtual or functional sphere. Even the emergence of Islamic contestation contributes to the consecration of the traditional repertory. On the other hand, the rise of world Islam during the last thirty years has opened up political opportunities for Morocco on the international stage – for instance the agencies of Islamic diplomacy (OCI, ISESCO or the quest for a new role in European Islam. By managing the interaction between the local and the global, the regime is able to keep itself in power.

  15. Versatile Cas9-Driven Subpopulation Selection Toolbox for Lactococcus lactis.

    Science.gov (United States)

    van der Els, Simon; James, Jennelle K; Kleerebezem, Michiel; Bron, Peter A

    2018-04-15

    CRISPR-Cas9 technology has been exploited for the removal or replacement of genetic elements in a wide range of prokaryotes and eukaryotes. Here, we describe the extension of the Cas9 application toolbox to the industrially important dairy species Lactococcus lactis The Cas9 expression vector pLABTarget, encoding the Streptocccus pyogenes Cas9 under the control of a constitutive promoter, was constructed, allowing plug and play introduction of short guide RNA (sgRNA) sequences to target specific genetic loci. Introduction of a pepN -targeting derivative of pLABTarget into L. lactis strain MG1363 led to a strong reduction in the number of transformants obtained, which did not occur in a pepN deletion derivative of the same strain, demonstrating the specificity and lethality of the Cas9-mediated double-strand breaks in the lactococcal chromosome. Moreover, the same pLABTarget derivative allowed the selection of a pepN deletion subpopulation from its corresponding single-crossover plasmid integrant precursor, accelerating the construction and selection of gene-specific deletion derivatives in L. lactis Finally, pLABTarget, which contained sgRNAs designed to target mobile genetic elements, allowed the effective curing of plasmids, prophages, and integrative conjugative elements (ICEs). These results establish that pLABTarget enables the effective exploitation of Cas9 targeting in L. lactis , while the broad-host-range vector used suggests that this toolbox could readily be expanded to other Gram-positive bacteria. IMPORTANCE Mobile genetic elements in Lactococcus lactis and other lactic acid bacteria (LAB) play an important role in dairy fermentation, having both positive and detrimental effects during the production of fermented dairy products. The pLABTarget vector offers an efficient cloning platform for Cas9 application in lactic acid bacteria. Targeting Cas9 toward mobile genetic elements enabled their effective curing, which is of particular interest in the

  16. L’optimisation juridique du paiement pour services environnementaux en faveur de la préservation des services environnementaux : le cas du Cameroun et de la République Démocratique du Congo

    Directory of Open Access Journals (Sweden)

    Blaise-Pascal Ntirumenyerwa Mihigo

    2017-02-01

    Full Text Available The study starts from a hypothesis on the coherence and compatibility of the legal instruments in force in Cameroon and in the Democratic Republic of Congo (DRC with the optimization of payment for environmental services (PES and the preservation of environmental services. This study has employed a legal approach and interviews in order to investigate whether there is coherence and compatibility or not between the two variables of this hypothesis: (1 the legal instruments in force in Cameroon and in the DRC and (2 the optimization of PES and the preservation of environmental services. This study consists of three parts. The first part deals with the theoretical framework of PES and the place of PES in the legal order. In this first part, the definition of an optimal PES, the indicators of an optimal PES and the categories of legal frameworks on PES have been revealed. These are the fundamental elements to conduct a systematic analysis in the second and third parts. Based on these fundamental elements, the study analyses the legal instru- ments from international, regional (Africa and domestic (Cameroon and the DRC levels and investigates through field research two PES projects, one in Cameroon called “PES comminatory project” and another in the DRC called “REDD CBFF-Luki” respectively in the second and the third parts. From the analysis of these legal instruments and the investigation of these two PES projects, it has become apparent that there is a lack of coherence and compatibility between the legal instruments and these two PES projects in Cameroon and in the DRC, and the optimization of PES and the preservation of environmental services in the Congo Basin in general, especially in these two States. Useful recommendations have been made to eradicate these shortcomings. Key words: payment for environmental services, environmental services, Congo Basin

  17. CAS course on Plasma Wake Acceleration

    CERN Multimedia

    CERN Accelerator School

    2015-01-01

    The CERN Accelerator School (CAS) recently organised a specialised course on Plasma Wake Acceleration, held at CERN, Geneva, Switzerland, from 23 to 29 November 2014.    Following a number of introductory lectures on laser and plasma physics, as well as an overview of conventional accelerators and their limitations, the course covered a large number of aspects of plasma wake acceleration schemes: the creation of plasma by high power lasers or particle beams, a description of the plasma creation process through simulations and the characteristics of the accelerated particle beams, including results of the latest achievements. Lectures on beam diagnostics, the applications of plasma accelerated beams, and topical seminars completed the programme.  The course was very successful, with 109 students of 26 nationalities attending; most participants coming from European counties, but also from the US, Israel, India, South Korea, Russia and Ukraine. Feedback from the participants was...

  18. CAS – A Journey Has Begun in Aotearoa New Zealand

    Directory of Open Access Journals (Sweden)

    Derek Smith

    2008-08-01

    Full Text Available This paper explores a journey through hand-held technology changes in mathematics teaching and learning and raises questions we as mathematics educators should be considering in the shorter and longer term. New Zealand is embarking on a Computer Algebraic Systems (CAS Pilot Programme in secondary school mathematics. The Ministry of Education and the New Zealand Qualifications Authority have selected secondary schools to be part of a pilot programme in the use of CAS technology in mathematics classes. The aim of the pilot programme is to improve teaching and learning of mathematics through the use of this technology. Six schools in 2005 used CAS technology with Year 9 (13-14 year olds students and, an additional 16 schools joined the programme in 2006. The pilot is planned to continue with an increasing number of schools in subsequent years. By the time students in the pilot schools reach Years 11, 12 and 13, alternative external assessments using the CAS technology will be available. Professional development support and assistance in obtaining and using the technology will be provided to the pilot schools. The project's emphasis in 2005 was on the Geometry and Algebra strands; the Statistics strand was added in 2006. By 2010 the first cohort of project programme students will have been through their secondary mathematics education via a CAS environment. New Zealand teachers have only a finite time to get into CAS technology and integrate it into their teaching practice. This paper discusses a research project based on a mathematics department professional development that is linked to the pilot.

  19. Enabling the maximum number of people to access essential services will not be possible without private sector involvement and appropriate pricing of the services concerned

    Directory of Open Access Journals (Sweden)

    Luc Rigouzzo

    2012-06-01

    Full Text Available Private sector provision of basic services (water, energy, financial services and housing for people in developing countries is a necessity if we really want to try to curb poverty. However, ‘traditional’ private funding is not spontaneously directed towards these sectors, largely as a result of rejecting the idea that poor population groups should ‘pay’ for essential services; an issue that has often been the subject of opposition campaigns mounted by social stakeholders. Nevertheless, there are many, many examples to show that given the impact of these services on their quality of life, consumers in these countries - and especially those at the ‘bottom of the pyramid’ - are prepared to pay for them as long as they have access to a high-quality service. In these sectors, the nominal cost of the service concerned matters much less than its opportunity cost and the impact it will have on the lives of those who benefit from it. Very often, this service may even be paid for in advance as a way of enabling families to gain greater control over the expenditure they can devote to obtaining it.It is, however, important to distinguish between the supply of essential services and those of consumer goods, and - of course - to avoid abuses. In any event, the possibility of building financially-balanced models is what governs the process of securing sufficient funding from local and international financial institutions. In this area, as in others, the way forward is probably the happy medium: avoiding the excesses of overpricing, but accepting the need to maintain profitable economic models. These should enable investors to receive a level of profit that encourages them to continue and increase their investment, thereby increasing the number of recipients as quickly as possible. Aspiring to build social models that reject the ambition to achieve a reasonable profit and rule out any distribution of dividends to shareholders is to condemn the

  20. Spacer capture and integration by a type I-F Cas1-Cas2-3 CRISPR adaptation complex.

    Science.gov (United States)

    Fagerlund, Robert D; Wilkinson, Max E; Klykov, Oleg; Barendregt, Arjan; Pearce, F Grant; Kieper, Sebastian N; Maxwell, Howard W R; Capolupo, Angela; Heck, Albert J R; Krause, Kurt L; Bostina, Mihnea; Scheltema, Richard A; Staals, Raymond H J; Fineran, Peter C

    2017-06-27

    CRISPR-Cas adaptive immune systems capture DNA fragments from invading bacteriophages and plasmids and integrate them as spacers into bacterial CRISPR arrays. In type I-E and II-A CRISPR-Cas systems, this adaptation process is driven by Cas1-Cas2 complexes. Type I-F systems, however, contain a unique fusion of Cas2, with the type I effector helicase and nuclease for invader destruction, Cas3. By using biochemical, structural, and biophysical methods, we present a structural model of the 400-kDa Cas1 4 -Cas2-3 2 complex from Pectobacterium atrosepticum with bound protospacer substrate DNA. Two Cas1 dimers assemble on a Cas2 domain dimeric core, which is flanked by two Cas3 domains forming a groove where the protospacer binds to Cas1-Cas2. We developed a sensitive in vitro assay and demonstrated that Cas1-Cas2-3 catalyzed spacer integration into CRISPR arrays. The integrase domain of Cas1 was necessary, whereas integration was independent of the helicase or nuclease activities of Cas3. Integration required at least partially duplex protospacers with free 3'-OH groups, and leader-proximal integration was stimulated by integration host factor. In a coupled capture and integration assay, Cas1-Cas2-3 processed and integrated protospacers independent of Cas3 activity. These results provide insight into the structure of protospacer-bound type I Cas1-Cas2-3 adaptation complexes and their integration mechanism.

  1. Expanding CRISPR/Cas9 Genome Editing Capacity in Zebrafish Using SaCas9

    OpenAIRE

    Feng, Yan; Chen, Cheng; Han, Yuxiang; Chen, Zelin; Lu, Xiaochan; Liang, Fang; Li, Song; Qin, Wei; Lin, Shuo

    2016-01-01

    The type II CRISPR/Cas9 system has been used widely for genome editing in zebrafish. However, the requirement for the 5′-NGG-3′ protospacer-adjacent motif (PAM) of Cas9 from Streptococcus pyogenes (SpCas9) limits its targeting sequences. Here, we report that a Cas9 ortholog from Staphylococcus aureus (SaCas9), and its KKH variant, successfully induced targeted mutagenesis with high frequency in zebrafish. Confirming previous findings, the SpCas9 variant, VQR, can also induce targeted mutation...

  2. Developing a case mix classification for child and adolescent mental health services: the influence of presenting problems, complexity factors and service providers on number of appointments.

    Science.gov (United States)

    Martin, Peter; Davies, Roger; Macdougall, Amy; Ritchie, Benjamin; Vostanis, Panos; Whale, Andy; Wolpert, Miranda

    2017-09-01

    Case-mix classification is a focus of international attention in considering how best to manage and fund services, by providing a basis for fairer comparison of resource utilization. Yet there is little evidence of the best ways to establish case mix for child and adolescent mental health services (CAMHS). To develop a case mix classification for CAMHS that is clinically meaningful and predictive of number of appointments attended and to investigate the influence of presenting problems, context and complexity factors and provider variation. We analysed 4573 completed episodes of outpatient care from 11 English CAMHS. Cluster analysis, regression trees and a conceptual classification based on clinical best practice guidelines were compared regarding their ability to predict number of appointments, using mixed effects negative binomial regression. The conceptual classification is clinically meaningful and did as well as data-driven classifications in accounting for number of appointments. There was little evidence for effects of complexity or context factors, with the possible exception of school attendance problems. Substantial variation in resource provision between providers was not explained well by case mix. The conceptually-derived classification merits further testing and development in the context of collaborative decision making.

  3. The number of service per conception of Indonesian Friesian Holstein with artificial insemination in Selo, Boyolali, Central Java, Indonesia

    Science.gov (United States)

    Wicaksono, A. M.; Pramono, A.; Susilowati, A.; Sutarno; Widyas, N.; Prastowo, S.

    2018-03-01

    Boyolali is an area in Central Java Indonesia, it has large number of Indonesian Friesian Holstein (IFH; dairy cattle). To improve its population as well as genetic quality of milk production, artificial insemination (AI) is widely applied as mating program. The success of AI can be evaluated from the number of service per conception (S/C), represent a number of service using AI to achieve one pregnancy. Its mirroring mating management and reproductive efficiency in dairy cattle, estimated in herd during specific time and location. For that, this study aims to estimate S/C in Selo, Boyolali during October 2016 to January 2017. Data were gathered with 95% confidence level. Sample size were 367 IFH, visited and selected purposively based on criteria one-time partus, 3 y.o and have complete AI record. Animal data were collected in reproduction and mating management. In addition, 124 dairy farmer who have minimum 5 years experiences in rearing IFH cow were interviewed as respondent in estrus detection, followed with 2 skilled inseminators for AI performing time data. Result shows that S/C is 1.71, this mean one pregnancy need 1.71 times AI services. In the estrus detection, most of dairy farmers were able to observe estrus sign in vulva color, size and the present of mucus by visual. Moreover, AI was performed in 9 to 12 hours after the sign of estrus observed. It is concluded that AI of IFH in Selo, Boyolali has been successfully applied, however there are still rooms to improve the reproduction efficiency through mating management in regard to lower S/C.

  4. Biophysical properties of intrinsically disordered p130Cas substrate domain--implication in mechanosensing.

    Directory of Open Access Journals (Sweden)

    Kinya Hotta

    2014-04-01

    Full Text Available Mechanical stretch-induced tyrosine phosphorylation in the proline-rich 306-residue substrate domain (CasSD of p130Cas (or BCAR1 has eluded an experimentally validated structural understanding. Cellular p130Cas tyrosine phosphorylation is shown to function in areas without internal actomyosin contractility, sensing force at the leading edge of cell migration. Circular dichroism shows CasSD is intrinsically disordered with dominant polyproline type II conformations. Strongly conserved in placental mammals, the proline-rich sequence exhibits a pseudo-repeat unit with variation hotspots 2-9 residues before substrate tyrosine residues. Atomic-force microscopy pulling experiments show CasSD requires minimal extension force and exhibits infrequent, random regions of weak stability. Proteolysis, light scattering and ultracentrifugation results show that a monomeric intrinsically disordered form persists for CasSD in solution with an expanded hydrodynamic radius. All-atom 3D conformer sampling with the TraDES package yields ensembles in agreement with experiment when coil-biased sampling is used, matching the experimental radius of gyration. Increasing β-sampling propensities increases the number of prolate conformers. Combining the results, we conclude that CasSD has no stable compact structure and is unlikely to efficiently autoinhibit phosphorylation. Taking into consideration the structural propensity of CasSD and the fact that it is known to bind to LIM domains, we propose a model of how CasSD and LIM domain family of transcription factor proteins may function together to regulate phosphorylation of CasSD and effect machanosensing.

  5. A p130Cas tyrosine phosphorylated substrate domain decoy disrupts v-Crk signaling

    Directory of Open Access Journals (Sweden)

    Hanafusa Hidesaburo

    2002-07-01

    Full Text Available Abstract Background The adaptor protein p130Cas (Cas has been shown to be involved in different cellular processes including cell adhesion, migration and transformation. This protein has a substrate domain with up to 15 tyrosines that are potential kinase substrates, able to serve as docking sites for proteins with SH2 or PTB domains. Cas interacts with focal adhesion plaques and is phosphorylated by the tyrosine kinases FAK and Src. A number of effector molecules have been shown to interact with Cas and play a role in its function, including c-crk and v-crk, two adaptor proteins involved in intracellular signaling. Cas function is dependent on tyrosine phosphorylation of its substrate domain, suggesting that tyrosine phosphorylation of Cas in part regulates its control of adhesion and migration. To determine whether the substrate domain alone when tyrosine phosphorylated could signal, we have constructed a chimeric Cas molecule that is phosphorylated independently of upstream signals. Results We found that a tyrosine phosphorylated Cas substrate domain acts as a dominant negative mutant by blocking Cas-mediated signaling events, including JNK activation by the oncogene v-crk in transient and stable lines and v-crk transformation. This block was the result of competition for binding partners as the chimera competed for binding to endogenous c-crk and exogenously expressed v-crk. Conclusion Our approach suggests a novel method to study adaptor proteins that require phosphorylation, and indicates that mere tyrosine phosphorylation of the substrate domain of Cas is not sufficient for its function.

  6. Les tuberculomes intracraniens: à propos de 125 cas

    Science.gov (United States)

    Moufid, Faycal; Oulali, Noureddine; El Fatemi, Nizare; Gana, Rachid; Maaqili, Rachid; Bellakhdar, Fouad

    2012-01-01

    Les tuberculomes intracrâniens représentent l'une des localisations les plus graves de la tuberculose, leur incidence varie en fonction du contexte représentant 0,2% des processus intracrâniens dans les pays occidentaux et 5 à 10% des masses intracrâniennes dans les pays en voie de développement. Nous rapportons une étude rétrospective de 125 cas. L'hypertension intracrânienne (45%) et le déficit neurologique (36%) sont les signes cliniques les plus fréquents. La lésion était localisée dans 60% des cas en sus-tentoriel et dans 40% des cas en sous-tentoriel. L'approche thérapeutique a consisté en un abord direct du tuberculome dans 67 cas (53%), une biopsie stéréotaxique dans 32 cas (25%), le traitement médical en première intention sans confirmation histologique dans 26 cas (20%). Avant 1993 notre service ne disposait pas de cadre de stéréotaxie, notre attitude thérapeutique consistait soit en un abord direct de la lésion dans 70% des cas, soit un traitement antituberculeux en première intention sans confirmation histologique (30%). Cette attitude était corrélée à une mortalité et morbidité non négligeables respectivement 3% et 10%. Après 1993; le taux d'abords direct a chuté a 38%, avec 47% de biopsies stéréotaxiques et seulement 13% des patients traités par antibacillaires sans preuve histologique. Ceci s'est accompagné d'une réduction significative de mortalité a 1,4% (p = 0,0003) et de morbidité a 2% (p = 0,0027). PMID:22937196

  7. 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.

  8. Spectral Time Series of the Cas A Supernova

    Science.gov (United States)

    Rest, Armin

    2016-10-01

    We propose to obtain time-resolved spectroscopy of the outburst of the enigmatic historical supernova Cas A using STIS spectroscopy of light scattered by a narrow filament of interstellar dust. Our group has identified recent, high-surface brightness filaments that are likely to provide high signal-to-noise reproduction of the evolving spectrum of the Cas A outburst using verified, published techniques developed by us.The timescales to see any appreciable evolution in individual astrophysical objects are typically many orders of magnitudes larger than a human life. As a result, astronomers study large numbers of objects at different stages of their evolution to connect how a single object should change with time. Cas A can provide us with the ability, to look back in time to the point of explosion by observing its light echoes - SN light scattered off of dust in the Milky Way, which causes a time delay in reaching us. In obtaining spectra of light echoes, we have been able to determine the maximum-light characteristics of the SN. Our goal here is to obtain a single STIS spectrum of a bright Cas A LE, which will provide us a time series of spectra and a spatially resolved light curve of the Cas A SN. With these data, we will measure the properties of the cooling envelope after the shock breakout of the SN to estimate the radius of the progenitor star. We will then be able to connect the progenitor star to the explosion to the SN to the SNR.

  9. SD-CAS: Spin Dynamics by Computer Algebra System.

    Science.gov (United States)

    Filip, Xenia; Filip, Claudiu

    2010-11-01

    A computer algebra tool for describing the Liouville-space quantum evolution of nuclear 1/2-spins is introduced and implemented within a computational framework named Spin Dynamics by Computer Algebra System (SD-CAS). A distinctive feature compared with numerical and previous computer algebra approaches to solving spin dynamics problems results from the fact that no matrix representation for spin operators is used in SD-CAS, which determines a full symbolic character to the performed computations. Spin correlations are stored in SD-CAS as four-entry nested lists of which size increases linearly with the number of spins into the system and are easily mapped into analytical expressions in terms of spin operator products. For the so defined SD-CAS spin correlations a set of specialized functions and procedures is introduced that are essential for implementing basic spin algebra operations, such as the spin operator products, commutators, and scalar products. They provide results in an abstract algebraic form: specific procedures to quantitatively evaluate such symbolic expressions with respect to the involved spin interaction parameters and experimental conditions are also discussed. Although the main focus in the present work is on laying the foundation for spin dynamics symbolic computation in NMR based on a non-matrix formalism, practical aspects are also considered throughout the theoretical development process. In particular, specific SD-CAS routines have been implemented using the YACAS computer algebra package (http://yacas.sourceforge.net), and their functionality was demonstrated on a few illustrative examples. Copyright © 2010 Elsevier Inc. All rights reserved.

  10. Semiotic and discursive variables in cas-based didactical engineering

    DEFF Research Database (Denmark)

    Winsløw, Carl

    2003-01-01

    CAS, didactical engeneering, Maple, semiotics, undergraduate teaching, mathematics, education, didactics......CAS, didactical engeneering, Maple, semiotics, undergraduate teaching, mathematics, education, didactics...

  11. RNA-guided transcriptional activation via CRISPR/dCas9 mimics overexpression phenotypes in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Jong-Jin Park

    Full Text Available Clustered regularly interspaced short palindromic repeats (CRISPR and the CRISPR associated protein 9 (Cas9 system allows effective gene modification through RNA-guided DNA targeting. The Cas9 has undergone a series of functional alterations from the original active endonuclease to partially or completely deactivated Cas9. The catalytically deactivated Cas9 (dCas9 offers a platform to regulate transcriptional expression with the addition of activator or repressor domains. We redesigned a CRISPR/Cas9 activation system by adding the p65 transactivating subunit of NF-kappa B and a heat-shock factor 1 (HSF activation domain to dCas9 bound with the VP64 (tetramer of VP16 activation domain for application in plants. The redesigned CRISPR/Cas9 activation system was tested in Arabidopsis to increase endogenous transcriptional levels of production of anthocyanin pigment 1 (PAP1 and Arabidopsis thaliana vacuolar H+-pyrophosphatase (AVP1. The expression of PAP1 was increased two- to three-fold and the activated plants exhibited purple leaves similar to that of PAP1 overexpressors. The AVP1 gene expression was increased two- to five-fold in transgenic plants. In comparison to the wild type, AVP1 activated plants had increased leaf numbers, larger single-leaf areas and improved tolerance to drought stress. The AVP1 activated plants showed similar phenotypes to AVP1 overexpressors. Therefore, the redesigned CRISPR/Cas9 activation system containing modified p65-HSF provides a simple approach for producing activated plants by upregulating endogenous transcriptional levels.

  12. CRISPR-Cas: Adapting to change.

    Science.gov (United States)

    Jackson, Simon A; McKenzie, Rebecca E; Fagerlund, Robert D; Kieper, Sebastian N; Fineran, Peter C; Brouns, Stan J J

    2017-04-07

    Bacteria and archaea are engaged in a constant arms race to defend against the ever-present threats of viruses and invasion by mobile genetic elements. The most flexible weapons in the prokaryotic defense arsenal are the CRISPR-Cas adaptive immune systems. These systems are capable of selective identification and neutralization of foreign DNA and/or RNA. CRISPR-Cas systems rely on stored genetic memories to facilitate target recognition. Thus, to keep pace with a changing pool of hostile invaders, the CRISPR memory banks must be regularly updated with new information through a process termed CRISPR adaptation. In this Review, we outline the recent advances in our understanding of the molecular mechanisms governing CRISPR adaptation. Specifically, the conserved protein machinery Cas1-Cas2 is the cornerstone of adaptive immunity in a range of diverse CRISPR-Cas systems. Copyright © 2017, American Association for the Advancement of Science.

  13. Using CRISPR-Cas systems as antimicrobials.

    Science.gov (United States)

    Bikard, David; Barrangou, Rodolphe

    2017-06-01

    Although CRISPR-Cas systems naturally evolved to provide adaptive immunity in bacteria and archaea, Cas nucleases can be co-opted to target chromosomal sequences rather than invasive genetic elements. Although genome editing is the primary outcome of self-targeting using CRISPR-based technologies in eukaryotes, self-targeting by CRISPR is typically lethal in bacteria. Here, we discuss how DNA damage introduced by Cas nucleases in bacteria can efficiently and specifically lead to plasmid curing or drive cell death. Specifically, we discuss how various CRISPR-Cas systems can be engineered and delivered using phages or phagemids as vectors. These principles establish CRISPR-Cas systems as potent and programmable antimicrobials, and open new avenues for the development of CRISPR-based tools for selective removal of bacterial pathogens and precise microbiome composition alteration. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference.

    Science.gov (United States)

    Hochstrasser, Megan L; Taylor, David W; Bhat, Prashant; Guegler, Chantal K; Sternberg, Samuel H; Nogales, Eva; Doudna, Jennifer A

    2014-05-06

    In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA-E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage.

  15. [Detection of CRSPR-Cas system in Streptococcus thermophiles].

    Science.gov (United States)

    Li, Wan; Liang, Hongzhang; Zhang, Danqing; Wang, Nana; Tang, Yaru; Li, Bailiang; Huo, Guicheng

    2016-04-14

    We aimed to detect the CRSPR-Cas system of six Streptococcus thermophilus. Bioinformatics method was used to predict CRSPR-Cas system of nine S. thermophilus that published in National Center for Biotechnology Information. Four primers were designed according to the flanking sequences of standard strains and the CRISPR-Cas system of six S. thermophilus have been detected by PCR method. S. thermophilus S4 had a Cas9 gene, others all had Cas9 gene, Cas10 gene and Cas9* gene. In addition, 79 and KLDS3.0207 still had Cas3 gene. Signature genes amplification of CRSPR-Cas system could predict the type of CRSPR-Cas system in unsequenced strains, these findings will help establish the foundation for the study of CRSPR-Cas system in lactic acid bacteria.

  16. Modification of CAS-protocol for improvement of security web-applications from unauthorized access

    Directory of Open Access Journals (Sweden)

    Alexey I Igorevich Alexandrov

    2017-07-01

    Full Text Available Dissemination of information technologies and the expansion of their application demand constantly increasing security level for users, operating with confidential information and personal data. The problem of setting up secure user identification is probably one of the most common tasks, which occur in the process of software development. Today, despite the availability of a large amount of authentication tools, new solutions, mechanisms and technologies are being introduced regularly. Primarily, it is done to increase the security level of data protection against unauthorized access. This article describes the experience of using central user authentication service based on CAS-protocol (CAS – Central Authentication Service and free open source software, analyzing its main advantages and disadvantages and describing the possibility of its modification, which would increase security of web-based information systems from being accessed illegally. The article contains recommendations for setting a maximum time limit for users working on services, integrated with central authentication; and, analyses the research of implementing modern web-technologies while using user authentication system based on CAS-protocol. In addition, it describes the ways of CAS-server modernization for developing additional modules: a module for collecting and analyzing the use of information systems, and another one, for a user management system. Furthermore, CAS-protocol can be used at universities and other organizations for creating a unified information environment in education.

  17. 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.

  18. CAS - CERN Accelerator School: Course on Digital Signal Processing

    CERN Document Server

    Digital Signal Processing; CAS 2007

    2008-01-01

    These proceedings present the lectures given at the twenty-first specialized course organized by the CERN Accelerator School (CAS), the topic being Digital Signal Processing. The course was held in Sigtuna, Sweden, from 31 May–9 June 2007. This is the first time this topic has been selected for a specialized course. Taking into account the number of related applications currently in use in accelerators around the world, it was recognized that such a topic should definitively be incorporated into the CAS series of specialized courses. The specific aim of the course was to introduce the participants to the use and programming of Digital Signal Processors (DSPs) and Field Programmable Gate Arrays (FPGAs) evaluation boards. The course consisted of lectures in the mornings covering fundamental background knowledge in mathematics, controls theory, design tools, programming hardware platforms, and implementation details. In the afternoons the students split into two groups with people working in pairs. One group w...

  19. CAS - CERN Accelerator School: Specialised course on Magnets

    CERN Document Server

    CAS 2009

    2010-01-01

    These proceedings present the lectures given at the twenty-third specialized course organized by the CERN Accelerator School (CAS), the topic being 'Magnets'. The course was held in Bruges, Belgium, from 16 to 25 June 2009. This is the first time this topic has been selected for a specialized course. Taking into account the number of related applications currently in use in accelerators around the world, but, even more important, the worrying decrease in the corresponding expertise in the different laboratories, it was recognized that such a topic should definitively be incorporated into the CAS series of specialized courses. The specific aim of the course was to introduce the participants to the basics of resistive magnet design and its underlying theoretical concepts. The first part of the school dealt with basic introductory courses such as Maxwell's equations for magnets, beam optics, physics and measurement of magnetic materials, the different types of resistive magnets and their respective performance, ...

  20. Fragmentation of the CRISPR-Cas Type I-B signature protein Cas8b.

    Science.gov (United States)

    Richter, Hagen; Rompf, Judith; Wiegel, Julia; Rau, Kristina; Randau, Lennart

    2017-11-01

    CRISPR arrays are transcribed into long precursor RNA species, which are further processed into mature CRISPR RNAs (crRNAs). Cas proteins utilize these crRNAs, which contain spacer sequences that can be derived from mobile genetic elements, to mediate immunity during a reoccurring virus infection. Type I CRISPR-Cas systems are defined by the presence of different Cascade interference complexes containing large and small subunits that play major roles during target DNA selection. Here, we produce the protein and crRNA components of the Type I-B CRISPR-Cas complex of Clostridium thermocellum and Methanococcus maripaludis. The C. thermocellum Cascade complexes were reconstituted and analyzed via size-exclusion chromatography. Activity of the heterologous M. maripaludis CRISPR-Cas system was followed using phage lambda plaques assays. The reconstituted Type-I-B Cascade complex contains Cas7, Cas5, Cas6b and the large subunit Cas8b. Cas6b can be omitted from the reconstitution protocol. The large subunit Cas8b was found to be represented by two tightly associated protein fragments and a small C-terminal Cas8b segment was identified in recombinant complexes and C. thermocellum cell lysate. Production of Cas8b generates a small C-terminal fragment, which is suggested to fulfill the role of the missing small subunit. A heterologous, synthetic M. maripaludis Type I-B system is active in E. coli against phage lambda, highlighting a potential for genome editing using endogenous Type-I-B CRISPR-Cas machineries. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. CAS at the Cockcroft Institute

    CERN Multimedia

    2007-01-01

    The CERN Accelerator School (CAS) and the UK’s new centre for accelerator science, the Cockcroft Institute, jointly organised a course on General Accelerator Physics, at intermediate level, at the Cockcroft Institute, Daresbury, UK, from 16 to 28 September 2007. The venue took advantage of the excellent new facilities in the Institute and the existing infrastructure of the adjacent Daresbury Laboratory. The intermediate level course followed established practice, with lectures on core topics in the mornings and specialised courses in the afternoons. The latter provided "hands-on" education and experience in the three selected topics: "RF Measurements Techniques", "Beam Instrumentation and Diagnostics" and "Optics Design and Correction". These proved to be highly successful, with participants choosing one course and following the topic throughout the school. Guided studies, tutorials, seminars and a poster session completed the prog...

  2. The effect of Philadelphia and Pennsylvania Clean Indoor Air Act on food services and drinking places sales and numbers, 1998-2011.

    Science.gov (United States)

    Ma, Zhen-Qiang; Fisher, Monica A

    2013-11-27

    Philadelphia enacted its Clean Indoor Air Act (CIAA) nearly 2 years before the statewide CIAA. In this study, we assessed the economic impact of CIAAs on 4 types of food services and drinking places and addressed the predominant limitation of previous pre-post ban studies, namely the lack of control for confounders and changes in secular trends over time. We analyzed data from Pennsylvania Department of Revenue Quarterly 1998-2011 taxable county-level revenue sales and number of food services and drinking places. Region-specific and type-specific adjusted sales and number of food services and drinking places accounted for consumer spending as a general economic indicator. Segmented regression analysis of interrupted time-series methodology assessed changes in trend and level. Pennsylvania CIAA had no significant effect on adjusted sales or numbers except for an increase in sales in Philadelphia for limited-service eating places and in the surrounding 4 counties for special food services. Philadelphia CIAA was associated with an increase in adjusted numbers of full-service restaurants in Philadelphia and the rest of the state, special food services in Philadelphia, and drinking places in the rest of the state, and a decrease in the number of special food services in the surrounding counties. Philadelphia CIAA had no significant effect on adjusted sales except for an increase in special food services in the rest of the state. Overall, CIAAs had no negative business-related impact and, for the most part, suggest a positive impact on restaurant sales and numbers. Our results provide further support for comprehensive CIAA ordinance for restaurants.

  3. Méningiome intracrânien multiple: expérience du service de neurochirurgie CHU Avicenne Rabat - Salé, à propos de 4 cas et revue de la literature

    Science.gov (United States)

    Djoubairou, Ben Ousmanou; Karekezi, Claire; Moussé, Nabil; Doleagbenou, Agbéko Komlan; Gana, Rachid; El Abbadi, Najia; El Maaqili, Moulay Rachid

    2014-01-01

    Les méningiomes intracrâniennes multiples sont définies comme la présence d'au moins deux méningiomes sur des sites intracrâniens différents et ceci en absence de neurofibromatose. C'est une tumeur rare dont la prévalence varie entre 1-10%. Le but de notre travail était de décrire les caractéristiques cliniques, radiologiques, histologiques d'une série de 4 patients porteurs de méningiome multiple et en déduire les facteurs de risques de survenue de cette pathologie. Préciser la qualité d'exérèse chirurgicale de la lésion selon la classification de Simpson. Rapporter les suites postopératoires ainsi que le suivie à long termes des patients afin de préciser leur qualité de vie. Il s'agit d'une étude rétrospective portant sur 4 cas de Méningiomes intracrâniens multiples sur 174 patients opérés pour méningiome au CHU Avicenne entre Janvier 2000 à Décembre 2013. En s'aidant des données cliniques, imageries, chirurgicales, histologiques mentionnée dans le dossier médical de chaque patient. Notre série est constitué de 4 patients (3 femmes pour 1 homme), d'un âge allant de 42-50 ans (moyenne d’âge= 45,5 ans). Nous avons identifié 21 méningiomes (17 en sus tentoriel et 4 en sous tentoriel), aucun cas de décès ni d'infection postopératoire dans notre échantillon. Le pronostic reste bon malgré le nombre de lésion nécessitant parfois plusieurs interventions chirurgicales. PMID:25419331

  4. 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.

  5. Parameters affecting frequency of CRISPR/Cas9 mediated targeted mutagenesis in rice.

    Science.gov (United States)

    Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

    2015-10-01

    Frequency of CRISPR/Cas9-mediated targeted mutagenesis varies depending on Cas9 expression level and culture period of rice callus. Recent reports have demonstrated that the CRISPR/Cas9 system can function as a sequence-specific nuclease in various plant species. Induction of mutation in proliferating tissue during embryogenesis or in germline cells is a practical means of generating heritable mutations. In the case of plant species in which cultured cells are used for transformation, non-chimeric plants can be obtained when regeneration occurs from mutated cells. Since plantlets are regenerated from both mutated and non-mutated cells in a random manner, any increment in the proportion of mutated cells in Cas9- and guide RNA (gRNA)-expressing cells will help increase the number of plants containing heritable mutations. In this study, we examined factors affecting mutation frequency in rice calli. Following sequential transformation of rice calli with Cas9- and gRNA- expression constructs, the mutation frequency in independent Cas9 transgenic lines was analyzed. A positive correlation between Cas9 expression level and mutation frequency was found. This positive relationship was observed regardless of whether the transgene or an endogenous gene was used as the target for CRISPR/Cas9-mediated mutagenesis. Furthermore, we found that extending the culture period increased the proportion of mutated cells as well as the variety of mutations obtained. Because mutated and non-mutated cells might proliferate equally, these results suggest that a prolonged tissue culture period increases the chance of inducing de novo mutations in non-mutated cells. This fundamental knowledge will help improve systems for obtaining non-chimeric regenerated plants in many plant species.

  6. A newly discovered Bordetella species carries a transcriptionally active CRISPR-Cas with a small Cas9 endonuclease

    Science.gov (United States)

    The Cas9 endonuclease of the Type II-a clustered regularly interspersed short palindromic repeats (CRISPR), of Streptococcus pyogenes (SpCas9) has been adapted as a widely used tool for genome editing and genome engineering. Herein, we describe a gene encoding a novel Cas9 ortholog (BpsuCas9) and th...

  7. Cas9 specifies functional viral targets during CRISPR-Cas adaptation.

    Science.gov (United States)

    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.

  8. A thermostable Cas9 with increased lifetime in human plasma

    OpenAIRE

    Harrington, LB; Paez-Espino, D; Staahl, BT; Chen, JS; Ma, E; Kyrpides, NC; Doudna, JA

    2017-01-01

    © 2017 The Author(s). CRISPR-Cas9 is a powerful technology that has enabled genome editing in a wide range of species. However, the currently developed Cas9 homologs all originate from mesophilic bacteria, making them susceptible to degradation and unsuitable for applications requiring cleavage at elevated temperatures. Here, we show that the Cas9 protein from the thermophilic bacterium Geobacillus stearothermophilus (GeoCas9) catalyzes RNA-guided DNA cleavage at elevated temperatures. GeoCas...

  9. AKRO/SF: Catch Accounting System (CAS)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The Catch Accounting System (CAS) creates total catch estimates for the groundfish fisheries in the Bering Sea/Aleutian Islands and Gulf of Alaska. Each year, quotas...

  10. The new CAS-DIS digital ionosonde

    Directory of Open Access Journals (Sweden)

    Wang Shun

    2013-04-01

    Full Text Available A high quality digital ionosonde called the Chinese Academy of Sciences digital ionosonde (CAS-DIS has been developed for investigations of the ionosphere. Two important features are used for the CAS-DIS; first, the technique of analog down-conversion has been replaced by the new approach of digital down-conversion technology. Secondly, to solve the problem of large instantaneous receiving bandwidth in digital receivers, an analog narrowband tracking filter is used for the CAS-DIS. The center frequency of the filter tracks the carrier frequency transmitted in real-time, to ensure that the frequency components are filtered out of the effective bandwidth. This report describes the system architecture of the CAS-DIS, its main features, and its test results for ionosphere detection. 

  11. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems

    Science.gov (United States)

    Fonfara, Ines; Le Rhun, Anaïs; Chylinski, Krzysztof; Makarova, Kira S.; Lécrivain, Anne-Laure; Bzdrenga, Janek; Koonin, Eugene V.; Charpentier, Emmanuelle

    2014-01-01

    The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA:crRNA duplex (dual-RNA), which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci. The reported collection of dual-RNA:Cas9 with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9 tool. PMID:24270795

  12. 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.

  13. 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

  14. Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

    International Nuclear Information System (INIS)

    Kennedy, Edward M.; Cullen, Bryan R.

    2015-01-01

    CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

  15. Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

    Energy Technology Data Exchange (ETDEWEB)

    Kennedy, Edward M.; Cullen, Bryan R., E-mail: bryan.cullen@duke.edu

    2015-05-15

    CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

  16. Fast Facts: Recent Statistics from the Library Research Service, Numbers 283-289. January-December, 2010

    Science.gov (United States)

    Library Research Service, 2010

    2010-01-01

    Issues 283 through 289 of "Fast Facts" from the Library Research Service present data collected from libraries in Colorado and throughout the nation. Topics addressed in these "Fast Facts" from 2010 include the relationship between computer access in libraries and use of traditional services, analysis of the third year of data…

  17. Assessment of Information and Communication Technology for Selective Dissemination of Information and Current Awareness Services: A Case Study of University Libraries in the South-West Zone of Nigeria

    Directory of Open Access Journals (Sweden)

    Saturday U. Omeluzor

    2017-12-01

    Full Text Available Abstract Objective – To assess the use of information and communication technology (ICT for selective dissemination of information (SDI and current awareness services (CAS in university libraries in the South-West zone of Nigeria. Methods – A descriptive research design was adopted. The instrument used for data collection was a structured questionnaire administered to a population consisting of 379 librarians, with 353 usable questionnaires retrieved. Results – Findings revealed that most university libraries in the South-West zone of Nigeria do not use ICT in delivery of SDI and CAS. It is evident in this study that despite the known positive effects of ICT in library services, traditional methods were predominantly used for SDI and CAS to the library users. The study revealed that erratic Internet services, insufficient training, inadequate ICT skills, and low support for ICT were hindrances towards ICT use for SDI and CAS. Conclusions – The integration of ICT features in library services for the delivery of CAS and SDI has been a challenge in university libraries in South-West Nigeria. Only a few libraries and a low percentage of librarians had adopted the use of ICT in the delivery of CAS and SDI, while a larger number of libraries resorted to the use of traditional methods. The level of ICT literacy among the librarians in this study is low, as a higher percentage of librarians did not have adequate ICT skill to use available online resources on the Internet and other ICT tools to deliver SDI and CAS in South-West, Nigeria. This is not unconnected to the fact that the training and technical support received by the librarians is inadequate, and the level of support that academic libraries received from their university managements in South-West Nigeria in terms of funding for ICT development is inadequate, which led to low Internet services.

  18. Alien Registration Number Verification via the U.S. Citizenship and Immigration Service's Systematic Alien Verification for Entitlements System

    National Research Council Canada - National Science Library

    Ainslie, Frances M; Buck, Kelly R

    2008-01-01

    The purpose of this study was to evaluate the implications of conducting high-volume automated checks of the United States Citizenship and Immigration Services Systematic Allen Verification for Entitlements System (SAVE...

  19. Improving the patient booking service to reduce the number of missed appointments at East London NHS Foundation Trust Community Musculoskeletal Physiotherapy Service.

    Science.gov (United States)

    Tan, Elizabeth; Shah, Amar; De Souza, Warren; Harrison, Mark; Chettur, Chris; Onathukattil, Maimoona; Smart, Michelle; Mata, Marlon; Chitewe, Auzewell; Binley, Emma

    2017-01-01

    The East London National Health Service Foundation Trust (ELFT) Community Musculoskeletal (MSK) Physiotherapy Service had reported a high rate of non-attendance at scheduled appointments. This was leading to delayed access to treatment for patients and a reduced capacity for service users, as well as a waste of clinical resources. The aim of this quality improvement project was therefore to reduce the percentage of missed appointments within this department. This study was undertaken by the ELFT community MSK service, with support from the ELFT Quality Improvement team. To begin with, patient complaints were explored; these indicated that the main reason for missing appointments was due to issues with the patient booking service. Baseline data were initially collected for both new referrals and follow-up patients. The proposed changes were then introduced, which included text message reminders, first via a manual platform and then via an automated system. Ongoing data were recorded to note the effectiveness of these changes. Following the intervention, non-attendance of newly referred patients reduced by 43.35% (23.76%-13.46%) after both cycles. Non-attendance of follow-up patients reduced by 44.14% (23.74%-13.26%) after the second cycle alone. By listening to the opinions of service users, it was possible to improve the patient booking system and the flexibility of appointments. This resulted in a reduction in the percentage of appointments missed. These changes will continue to be monitored within this department to ensure sustainability but there is also now potential for similar interventions to be trialled in other health service departments.

  20. Cytosolic and Nuclear Delivery of CRISPR/Cas9-ribonucleoprotein for Gene Editing Using Arginine Functionalized Gold Nanoparticles.

    Science.gov (United States)

    Mout, Rubul; Rotello, Vincent M

    2017-10-20

    In this protocol, engineered Cas9-ribonucleoprotein (Cas9 protein and sgRNA, together called Cas9-RNP) and gold nanoparticles are used to make nanoassemblies that are employed to deliver Cas9-RNP into cell cytoplasm and nucleus. Cas9 protein is engineered with an N-terminus glutamic acid tag (E-tag or En, where n = the number of glutamic acid in an E-tag and usually n = 15 or 20), C-terminus nuclear localizing signal (NLS), and a C-terminus 6xHis-tag. [Cas9En hereafter] To use this protocol, the first step is to generate the required materials (gold nanoparticles, recombinant Cas9En, and sgRNA). Laboratory-synthesis of gold nanoparticles can take up to a few weeks, but can be synthesized in large batches that can be used for many years without compromising the quality. Cas9En can be cloned from a regular SpCas9 gene (Addgene plasmid id = 47327), and expressed and purified using standard laboratory procedures which are not a part of this protocol. Similarly, sgRNA can be laboratory-synthesized using in vitro transcription from a template gene (Addgene plasmid id = 51765) or can be purchased from various sources. Once these materials are ready, it takes about ~30 min to make the Cas9En-RNP complex and 10 min to make the Cas9En-RNP/nanoparticles nanoassemblies, which are immediately used for delivery (Figure 1). Complete delivery (90-95% cytoplasmic and nuclear delivery) is achieved in less than 3 h. Follow-up editing experiments require additional time based on users' need. Synthesis of arginine functionalized gold nanoparticles (ArgNPs) (Yang et al ., 2011), expression of recombinant Cas9En, and in vitro synthesis of sgRNA is reported elsewhere (Mout et al ., 2017). We report here only the generation of the delivery vehicle i.e. , the fabrication of Cas9En-RNP/ArgNPs nanoassembly.

  1. Procuring Contracting Officers’ Perceptions of the Contributions Made to Defense Cost Accounting Practices by CAS 401-416.

    Science.gov (United States)

    1981-09-01

    8217 PERCEPTIONS OF THE CONTRIBUTIONS MADE TO DEFENSE COST ACCOUNTING PRACTICES BY CAS 401-416 Captain Bruce E. Simpson, USA LSSR 70-81 The contents of...CONTRIBUTIONS MADE TO DEFENSE MastersThesis COST ACCOUNTING PRACTICES BY CAS 401-416 6. PEROR ING OG. REPORT NUMBER 7. AUTHOR(e) S. CONTRACT OR GRANT...SUPPLEMENTARY NOTES 19. KEY WORDS (Con~tiue, on revere side it naoeaaeuy and Identify by block nuffler) Accounting Cost Accounting Cost Accounting Standards

  2. A Broad-Spectrum Inhibitor of CRISPR-Cas9.

    Science.gov (United States)

    Harrington, Lucas B; Doxzen, Kevin W; Ma, Enbo; Liu, Jun-Jie; Knott, Gavin J; Edraki, Alireza; Garcia, Bianca; Amrani, Nadia; Chen, Janice S; Cofsky, Joshua C; Kranzusch, Philip J; Sontheimer, Erik J; Davidson, Alan R; Maxwell, Karen L; Doudna, Jennifer A

    2017-09-07

    CRISPR-Cas9 proteins function within bacterial immune systems to target and destroy invasive DNA and have been harnessed as a robust technology for genome editing. Small bacteriophage-encoded anti-CRISPR proteins (Acrs) can inactivate Cas9, providing an efficient off switch for Cas9-based applications. Here, we show that two Acrs, AcrIIC1 and AcrIIC3, inhibit Cas9 by distinct strategies. AcrIIC1 is a broad-spectrum Cas9 inhibitor that prevents DNA cutting by multiple divergent Cas9 orthologs through direct binding to the conserved HNH catalytic domain of Cas9. A crystal structure of an AcrIIC1-Cas9 HNH domain complex shows how AcrIIC1 traps Cas9 in a DNA-bound but catalytically inactive state. By contrast, AcrIIC3 blocks activity of a single Cas9 ortholog and induces Cas9 dimerization while preventing binding to the target DNA. These two orthogonal mechanisms allow for separate control of Cas9 target binding and cleavage and suggest applications to allow DNA binding while preventing DNA cutting by Cas9. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. A description of assistive technology sources, services and outcomes of use in a number of African settings.

    Science.gov (United States)

    Visagie, Surona; Eide, Arne H; Mannan, Hasheem; Schneider, Marguerite; Swartz, Leslie; Mji, Gubela; Munthali, Alister; Khogali, Mustafa; van Rooy, Gert; Hem, Karl-Gerhard; MacLachlan, Malcolm

    2017-10-01

    Purpose statement: The article explores assistive technology sources, services and outcomes in South Africa, Namibia, Malawi and Sudan. A survey was done in purposively selected sites of the study countries. Cluster sampling followed by random sampling served to identify 400-500 households (HHs) with members with disabilities per country. A HH questionnaire and individual questionnaire was completed. Country level analysis was limited to descriptive statistics. Walking mobility aids was most commonly bought/provided (46.3%), followed by visual aids (42.6%). The most common sources for assistive technology were government health services (37.8%), "other" (29.8%), and private health services (22.9%). Out of the participants, 59.3% received full information in how to use the device. Maintenance was mostly done by users and their families (37.3%). Devices helped a lot in 73.3% of cases and improved quality of life for 67.9% of participants, while 39.1% experienced functional difficulties despite the devices. Although there is variation between the study settings, the main impression is that of fragmented or absent systems of provision of assistive technology. Implications for rehabilitation Provision of assistive technology and services varied between countries, but the overall impression was of poor provision and fragmented services. The limited provision of assistive technology for personal care and handling products is of concern as many of these devices requires little training and ongoing support while they can make big functional differences. Rural respondents experienced more difficulties when using the device and received less information on use and maintenance of the device than their urban counterparts. A lack of government responsibility for assistive device services correlated with a lack of information and/or training of participants and maintenance of devices.

  4. 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

  5. CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles.

    Science.gov (United States)

    Lemos, Brenda R; Kaplan, Adam C; Bae, Ji Eun; Ferrazzoli, Alexander E; Kuo, James; Anand, Ranjith P; Waterman, David P; Haber, James E

    2018-02-27

    Harnessing CRISPR-Cas9 technology provides an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in budding yeast and found that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a 1-bp (+1) insertion that appears to result from Cas9 creating a 1-nt 5' overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which are biased toward homonucleotide insertions. Using inverted PAM sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9:gRNA complex influences subsequent NHEJ processing. As with events induced by the site-specific HO endonuclease, CRISPR-Cas9-mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11's nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

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

    Science.gov (United States)

    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.

  7. CRISPR/Cas9 system and its applications in human hematopoietic cells.

    Science.gov (United States)

    Hu, Xiaotang

    2016-11-01

    Since 2012, the CRISPR-Cas9 system has been quickly and successfully tested in a broad range of organisms and cells including hematopoietic cells. The application of CRISPR-Cas9 in human hematopoietic cells mainly involves the genes responsible for HIV infection, β-thalassemia and sickle cell disease (SCD). The successful disruption of CCR5 and CXCR4 genes in T cells by CRISPR-Cas9 promotes the prospect of the technology in the functional cure of HIV. More recently, eliminating CCR5 and CXCR4 in induced pluripotent stem cells (iPSCs) derived from patients and targeting the HIV genome have been successfully carried out in several laboratories. The outcome from these approaches bring us closer to the goal of eradicating HIV infection. For hemoglobinopathies the ability to produce iPSC-derived from patients with the correction of hemoglobin (HBB) mutations by CRISPR-Cas9 has been tested in a number of laboratories. These corrected iPSCs also show the potential to differentiate into mature erythrocytes expressing high-level and normal HBB. In light of the initial success of CRESPR-Cas9 in target mutated gene(s) in the iPSCs, a combination of genomic editing and autogenetic stem cell transplantation would be the best strategy for root treatment of the diseases, which could replace traditional allogeneic stem cell transplantation. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Numbers, systems, people: how interactions influence integration. Insights from case studies of HIV and reproductive health services delivery in Kenya.

    Science.gov (United States)

    Mayhew, Susannah H; Sweeney, Sedona; Warren, Charlotte E; Collumbien, Martine; Ndwiga, Charity; Mutemwa, Richard; Lut, Irina; Colombini, Manuela; Vassall, Anna

    2017-11-01

    Drawing on rich data from the Integra evaluation of integrated HIV and reproductive-health services, we explored the interaction of systems hardware and software factors to explain why some facilities were able to implement and sustain integrated service delivery while others were not. This article draws on detailed mixed-methods data for four case-study facilities offering reproductive-health and HIV services between 2009 and 2013 in Kenya: (i) time-series client flow, tracking service uptake for 8841 clients; (ii) structured questionnaires with 24 providers; (iii) in-depth interviews with 17 providers; (iv) workload and facility data using a periodic activity review and cost-instruments; and (v) contextual data on external activities related to integration in study sites. Overall, our findings suggested that although structural factors like stock-outs, distribution of staffing and workload, rotation of staff can affect how integrated care is provided, all these factors can be influenced by staff themselves: both frontline and management. Facilities where staff displayed agency of decision making, worked as a team to share workload and had management that supported this, showed better integration delivery and staff were able to overcome some structural deficiencies to enable integrated care. Poor-performing facilities had good structural integration, but staff were unable to utilize this because they were poorly organized, unsupported or teams were dysfunctional. Conscientious objection and moralistic attitudes were also barriers.Integra has demonstrated that structural integration is not sufficient for integrated service delivery. Rather, our case studies show that in some cases excellent leadership and peer-teamwork enabled facilities to perform well despite resource shortages. The ability to provide support for staff to work flexibly to deliver integrated services and build resilient health systems to meet changing needs is particularly relevant as health

  9. Engineered CRISPR-Cas9 nucleases with altered PAM specificities.

    Science.gov (United States)

    Kleinstiver, Benjamin P; Prew, Michelle S; Tsai, Shengdar Q; Topkar, Ved V; Nguyen, Nhu T; Zheng, Zongli; Gonzales, Andrew P W; Li, Zhuyun; Peterson, Randall T; Yeh, Jing-Ruey Joanna; Aryee, Martin J; Joung, J Keith

    2015-07-23

    Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.

  10. Report of the Hydrographic Service Royal Australian Navy for the Year Ended 30th June 1983, Issue Number 19.

    Science.gov (United States)

    1983-01-01

    eastwards of MORESBY’s 1981 survey. The ship suffered considerable problems in establishing her ARGO stations on this barren and inhospitable coast...on 6 months exchange whilst an RFMF officer pined experience in Australia. A CPOSR has continued the loan service arrang-ments in support of the...training in hydrographic surveying is undertaken at the Royal Navy’s Hydrographic School in Plymouth . It is expected that an average of four officers will

  11. An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes.

    Directory of Open Access Journals (Sweden)

    Simon E Tröder

    Full Text Available Electroporation of zygotes represents a rapid alternative to the elaborate pronuclear injection procedure for CRISPR/Cas9-mediated genome editing in mice. However, current protocols for electroporation either require the investment in specialized electroporators or corrosive pre-treatment of zygotes which compromises embryo viability. Here, we describe an easily adaptable approach for the introduction of specific mutations in C57BL/6 mice by electroporation of intact zygotes using a common electroporator with synthetic CRISPR/Cas9 components and minimal technical requirement. Direct comparison to conventional pronuclear injection demonstrates significantly reduced physical damage and thus improved embryo development with successful genome editing in up to 100% of living offspring. Hence, our novel approach for Easy Electroporation of Zygotes (EEZy allows highly efficient generation of CRISPR/Cas9 transgenic mice while reducing the numbers of animals required.

  12. Versatile Cas9-driven subpopulation selection toolbox for Lactococcus lactis

    NARCIS (Netherlands)

    Els, van der Simon; James, Jennelle K.; Kleerebezem, Michiel; Bron, Peter A.

    2018-01-01

    CRISPR-Cas9 technology has been exploited for the removal or replacement of genetic elements in a wide range of prokaryotes and eukaryotes. Here, we describe the extension of the Cas9 application toolbox to the industrially important dairy species Lactococcus lactis. The Cas9 expression vector

  13. Rational Design of Mini-Cas9 for Transcriptional Activation.

    Science.gov (United States)

    Ma, Dacheng; Peng, Shuguang; Huang, Weiren; Cai, Zhiming; Xie, Zhen

    2018-04-20

    Nuclease dead Cas9 (dCas9) has been widely used for modulating gene expression by fusing with different activation or repression domains. However, delivery of the CRISPR/Cas system fused with various effector domains in a single adeno-associated virus (AAV) remains challenging due to the payload limit. Here, we engineered a set of downsized variants of Cas9 including Staphylococcus aureus Cas9 (SaCas9) that retained DNA binding activity by deleting conserved functional domains. We demonstrated that fusing FokI nuclease domain to the N-terminal of the minimal SaCas9 (mini-SaCas9) or to the middle of the split mini-SaCas9 can trigger efficient DNA cleavage. In addition, we constructed a set of compact transactivation domains based on the tripartite VPR activation domain and self-assembled arrays of split SpyTag:SpyCatch peptides, which are suitable for fusing to the mini-SaCas9. Lastly, we produced a single AAV containing the mini-SaCas9 fused with a downsized transactivation domain along with an optimized gRNA expression cassette, which showed efficient transactivation activity. Our results highlighted a practical approach to generate down-sized CRISPR/Cas9 and gene activation systems for in vivo applications.

  14. Hernie de Spiegel: a propos d’un cas

    Directory of Open Access Journals (Sweden)

    Karim Ibn Majdoub Hassani

    2010-02-01

    Full Text Available La hernie de Spiegel ou hernie ventrale latérale est une déhiscence inhabituelle apparaissant sur la ligne ou fascia semi-lunaire de Spiegel. C’est une entité clinique rare, représente 0.10 à 1 pourcent des hernies. Aussi, nous a-t-il paru opportun de rapporter ce cas colligé dans le service de chirurgie B du CHU Hassan II de Fès. Nous rapportons l’observation d’une patiente âgée de 60 ans, sans antécédent particulier qui présentais une tuméfaction para ombilicale gauche augmentant progressivement de volume, Une hernie de Spiegel a été suspectée à l’examen clinique, et le diagnostic d’éventration antérolatérale gauche a été retenu à la tomodensitométrie abdominale. Une cure de la hernie par plaque de prolène a été réalisée et les suites opératoires étaient simples. La hernie de Spiegel est une affection rare, son diagnostic clinique peut être difficile. Elle est asymptomatique dans 90 pourcent des cas et Son diagnostic positif est radiologique. Le risque d’étranglement non négligeable impose un traitement chirurgical une fois le diagnostic est confirmé.

  15. Boosting plant immunity with CRISPR/Cas

    OpenAIRE

    Chaparro-Garcia, Angela; Kamoun, Sophien; Nekrasov, Vladimir

    2015-01-01

    CRISPR/Cas has recently been transferred to plants to make them resistant to geminiviruses, a damaging family of DNA viruses. We discuss the potential and the limitations of this method. See related Research: http://www.genomebiology.com/2015/16/1/238

  16. CRISPR-Cas : Adapting to change

    NARCIS (Netherlands)

    Jackson, Simon A.; McKenzie, R.E.; Fagerlund, Robert D.; Kieper, S.N.; Fineran, Peter C.; Brouns, S.J.J.

    2017-01-01

    Bacteria and archaea are engaged in a constant arms race to defend against the ever-present threats of viruses and invasion by mobile genetic elements. The most flexible weapons in the prokaryotic defense arsenal are the CRISPR-Cas adaptive immune systems. These systems are capable of selective

  17. 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

  18. 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...

  19. Cas9 versus Cas12a/Cpf1: Structure-function comparisons and implications for genome editing.

    Science.gov (United States)

    Swarts, Daan C; Jinek, Martin

    2018-05-22

    Cas9 and Cas12a are multidomain CRISPR-associated nucleases that can be programmed with a guide RNA to bind and cleave complementary DNA targets. The guide RNA sequence can be varied, making these effector enzymes versatile tools for genome editing and gene regulation applications. While Cas9 is currently the best-characterized and most widely used nuclease for such purposes, Cas12a (previously named Cpf1) has recently emerged as an alternative for Cas9. Cas9 and Cas12a have distinct evolutionary origins and exhibit different structural architectures, resulting in distinct molecular mechanisms. Here we compare the structural and mechanistic features that distinguish Cas9 and Cas12a, and describe how these features modulate their activity. We discuss implications for genome editing, and how they may influence the choice of Cas9 or Cas12a for specific applications. Finally, we review recent studies in which Cas12a has been utilized as a genome editing tool. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes. © 2018 Wiley Periodicals, Inc.

  20. La prise en compte des innovations en matière de mobilité dans la planification urbaine : le cas des Services de transports personnalisés (STP)

    OpenAIRE

    Castex, Élodie; Frère, Séverine; Groux, Annette

    2017-01-01

    Cet article s’intéresse à la façon dont la planification peut ou non permettre aux politiques de déplacements d’innover, de prendre en compte des nouveaux besoins et formes de mobilité. S’appuyant sur un travail de recherche qui porte sur la place des modes de transports alternatifs que sont le covoiturage, l’autopartage, le vélo-en-libre-service et le transport à la demande dans les politiques publiques, cet article tentera de comprendre comment les modes de déplacement innovants sont pris e...

  1. Simple and Efficient Targeting of Multiple Genes Through CRISPR-Cas9 in Physcomitrella patens

    Directory of Open Access Journals (Sweden)

    Mauricio Lopez-Obando

    2016-11-01

    Full Text Available Powerful genome editing technologies are needed for efficient gene function analysis. The CRISPR-Cas9 system has been adapted as an efficient gene-knock-out technology in a variety of species. However, in a number of situations, knocking out or modifying a single gene is not sufficient; this is particularly true for genes belonging to a common family, or for genes showing redundant functions. Like many plants, the model organism Physcomitrella patens has experienced multiple events of polyploidization during evolution that has resulted in a number of families of duplicated genes. Here, we report a robust CRISPR-Cas9 system, based on the codelivery of a CAS9 expressing cassette, multiple sgRNA vectors, and a cassette for transient transformation selection, for gene knock-out in multiple gene families. We demonstrate that CRISPR-Cas9-mediated targeting of five different genes allows the selection of a quintuple mutant, and all possible subcombinations of mutants, in one experiment, with no mutations detected in potential off-target sequences. Furthermore, we confirmed the observation that the presence of repeats in the vicinity of the cutting region favors deletion due to the alternative end joining pathway, for which induced frameshift mutations can be potentially predicted. Because the number of multiple gene families in Physcomitrella is substantial, this tool opens new perspectives to study the role of expanded gene families in the colonization of land by plants.

  2. Desserte ferroviaire à grande vitesse, activation des ressources spécifiques et développement du tourisme : le cas de l’agglomération rémoise High speed rail service, specific resources activation, and tourist development: the case of Rheims

    Directory of Open Access Journals (Sweden)

    Sylvie Bazin

    2012-12-01

    Full Text Available La LGV est-européenne a été mise en service le 10 juin 2007. Les acteurs économiques des territoires desservis attendent beaucoup de cette arrivée notamment en matière de développement des tourismes urbain et d’affaires. Mais un tel développement n’est pas automatique. C’est l’appropriation collective de l’innovation que constitue la desserte à grande vitesse dans un territoire qui est au coeur de ses effets positifs. En effet, cette appropriation collective constitue une innovation relationnelle de laquelle peuvent naître des innovations de services complémentaires en matière de tourisme susceptibles de valoriser les ressources spécifiques existantes (patrimoine historique, culturel, gastronomique, etc.. Ainsi, si l’existence de ressources spécifiques joue un rôle pour les villes desservies, les efforts coordonnés des acteurs pour favoriser la “mise en tourisme” de la ville sont également décisifs. L’analyse est illustrée par le cas de l’agglomération rémoise.The East European High-Speed Rail started on June 10, 2007. Economic actors of beneficiary territories are expecting a lot of positive effects such as the development of urban and business tourisms. But such development is not automatic. The collective appropriation of the innovations linked to a High-Speed Rail Service seems to be central for generating positive effects. Indeed, this collective appropriation constitutes a relational innovation, which may give birth to additional services innovations in tourism that could enhance the value of existing specific resources (historical, cultural, gastronomic, etc. and turn them into assets. Thus, if the availability of specific resources plays a specific role for the connected cities, coordinated efforts of actors to promote the tourist development of the city are also decisive. We will illustrate our subject with the case of Rheims.

  3. Increasing the Number of Outpatients Receiving Spiritual Assessment: A Pain and Palliative Care Service Quality Improvement Project.

    Science.gov (United States)

    Gomez-Castillo, Blanca J; Hirsch, Rosemarie; Groninger, Hunter; Baker, Karen; Cheng, M Jennifer; Phillips, Jayne; Pollack, John; Berger, Ann M

    2015-11-01

    Spirituality is a patient need that requires special attention from the Pain and Palliative Care Service team. This quality improvement project aimed to provide spiritual assessment for all new outpatients with serious life-altering illnesses. Percentage of new outpatients receiving spiritual assessment (Faith, Importance/Influence, Community, Address/Action in care, psychosocial evaluation, chaplain consults) at baseline and postinterventions. Interventions included encouraging clinicians to incorporate adequate spiritual assessment into patient care and implementing chaplain covisits for all initial outpatient visits. The quality improvement interventions increased spiritual assessment (baseline vs. postinterventions): chaplain covisits (25.5% vs. 50%), Faith, Importance/Influence, Community, Address/Action in care completion (49% vs. 72%), and psychosocial evaluation (89% vs. 94%). Improved spiritual assessment in an outpatient palliative care clinic setting can occur with a multidisciplinary approach. This project also identifies data collection and documentation processes that can be targeted for improvement. Published by Elsevier Inc.

  4. On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires.

    Science.gov (United States)

    Silas, Sukrit; Makarova, Kira S; Shmakov, Sergey; Páez-Espino, David; Mohr, Georg; Liu, Yi; Davison, Michelle; Roux, Simon; Krishnamurthy, Siddharth R; Fu, Becky Xu Hua; Hansen, Loren L; Wang, David; Sullivan, Matthew B; Millard, Andrew; Clokie, Martha R; Bhaya, Devaki; Lambowitz, Alan M; Kyrpides, Nikos C; Koonin, Eugene V; Fire, Andrew Z

    2017-07-11

    analysis of available bacterial sequence data, we find evidence that RT-based RNA adaptation machinery has been able to join with CRISPR-Cas immune systems in many, diverse bacterial species. To investigate whether the abilities to adapt to DNA and RNA molecules are utilized for defense against distinct classes of invaders in nature, we sequenced CRISPR arrays from samples of commercial-scale open-air cultures of Arthrospira platensis , a cyanobacterium that contains both RT-lacking and RT-containing CRISPR-Cas systems. We uncovered a diverse pool of naturally occurring immune memories, with the RT-lacking locus acquiring a number of segments matching known viral or bacterial genes, while the RT-containing locus has acquired spacers from a distinct sequence pool for which the source remains enigmatic. Copyright © 2017 Silas et al.

  5. Naturally Occurring Off-Switches for CRISPR-Cas9.

    Science.gov (United States)

    Pawluk, April; Amrani, Nadia; Zhang, Yan; Garcia, Bianca; Hidalgo-Reyes, Yurima; Lee, Jooyoung; Edraki, Alireza; Shah, Megha; Sontheimer, Erik J; Maxwell, Karen L; Davidson, Alan R

    2016-12-15

    CRISPR-Cas9 technology would be enhanced by the ability to inhibit Cas9 function spatially, temporally, or conditionally. Previously, we discovered small proteins encoded by bacteriophages that inhibit the CRISPR-Cas systems of their host bacteria. These "anti-CRISPRs" were specific to type I CRISPR-Cas systems that do not employ the Cas9 protein. We posited that nature would also yield Cas9 inhibitors in response to the evolutionary arms race between bacteriophages and their hosts. Here, we report the discovery of three distinct families of anti-CRISPRs that specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis. We show that these proteins bind directly to N. meningitidis Cas9 (NmeCas9) and can be used as potent inhibitors of genome editing by this system in human cells. These anti-CRISPR proteins now enable "off-switches" for CRISPR-Cas9 activity and provide a genetically encodable means to inhibit CRISPR-Cas9 genome editing in eukaryotes. VIDEO ABSTRACT. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. 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.

  7. Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems

    Directory of Open Access Journals (Sweden)

    Wolf Yuri I

    2011-07-01

    Full Text Available Abstract Background The CRISPR-Cas adaptive immunity systems that are present in most Archaea and many Bacteria function by incorporating fragments of alien genomes into specific genomic loci, transcribing the inserts and using the transcripts as guide RNAs to destroy the genome of the cognate virus or plasmid. This RNA interference-like immune response is mediated by numerous, diverse and rapidly evolving Cas (CRISPR-associated proteins, several of which form the Cascade complex involved in the processing of CRISPR transcripts and cleavage of the target DNA. Comparative analysis of the Cas protein sequences and structures led to the classification of the CRISPR-Cas systems into three Types (I, II and III. Results A detailed comparison of the available sequences and structures of Cas proteins revealed several unnoticed homologous relationships. The Repeat-Associated Mysterious Proteins (RAMPs containing a distinct form of the RNA Recognition Motif (RRM domain, which are major components of the CRISPR-Cas systems, were classified into three large groups, Cas5, Cas6 and Cas7. Each of these groups includes many previously uncharacterized proteins now shown to adopt the RAMP structure. Evidence is presented that large subunits contained in most of the CRISPR-Cas systems could be homologous to Cas10 proteins which contain a polymerase-like Palm domain and are predicted to be enzymatically active in Type III CRISPR-Cas systems but inactivated in Type I systems. These findings, the fact that the CRISPR polymerases, RAMPs and Cas2 all contain core RRM domains, and distinct gene arrangements in the three types of CRISPR-Cas systems together provide for a simple scenario for origin and evolution of the CRISPR-Cas machinery. Under this scenario, the CRISPR-Cas system originated in thermophilic Archaea and subsequently spread horizontally among prokaryotes. Conclusions Because of the extreme diversity of CRISPR-Cas systems, in-depth sequence and structure

  8. Changes in monthly unemployment rates may predict changes in the number of psychiatric presentations to emergency services in South Australia.

    Science.gov (United States)

    Bidargaddi, Niranjan; Bastiampillai, Tarun; Schrader, Geoffrey; Adams, Robert; Piantadosi, Cynthia; Strobel, Jörg; Tucker, Graeme; Allison, Stephen

    2015-07-24

    To determine the extent to which variations in monthly Mental Health Emergency Department (MHED) presentations in South Australian Public Hospitals are associated with the Australian Bureau of Statistics (ABS) monthly unemployment rates. Times series modelling of relationships between monthly MHED presentations to South Australian Public Hospitals derived from the Integrated South Australian Activity Collection (ISAAC) data base and the ABS monthly unemployment rates in South Australia between January 2004-June 2011. Time series modelling using monthly unemployment rates from ABS as a predictor variable explains 69% of the variation in monthly MHED presentations across public hospitals in South Australia. Thirty-two percent of the variation in current month's male MHED presentations can be predicted by using the 2 months' prior male unemployment rate. Over 63% of the variation in monthly female MHED presentations can be predicted by either male or female prior monthly unemployment rates. The findings of this study highlight that even with the relatively favourable economic conditions, small shifts in monthly unemployment rates can predict variations in monthly MHED presentations, particularly for women. Monthly ABS unemployment rates may be a useful metric for predicting demand for emergency mental health services.

  9. Rapid, Selection-Free, High-Efficiency Genome Editing in Protozoan Parasites Using CRISPR-Cas9 Ribonucleoproteins

    Directory of Open Access Journals (Sweden)

    Lia Carolina Soares Medeiros

    2017-11-01

    Full Text Available Trypanosomatids (order Kinetoplastida, including the human pathogens Trypanosoma cruzi (agent of Chagas disease, Trypanosoma brucei, (African sleeping sickness, and Leishmania (leishmaniasis, affect millions of people and animals globally. T. cruzi is considered one of the least studied and most poorly understood tropical disease-causing parasites, in part because of the relative lack of facile genetic engineering tools. This situation has improved recently through the application of clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 9 (CRISPR-Cas9 technology, but a number of limitations remain, including the toxicity of continuous Cas9 expression and the long drug marker selection times. In this study, we show that the delivery of ribonucleoprotein (RNP complexes composed of recombinant Cas9 from Staphylococcus aureus (SaCas9, but not from the more routinely used Streptococcus pyogenes Cas9 (SpCas9, and in vitro-transcribed single guide RNAs (sgRNAs results in rapid gene edits in T. cruzi and other kinetoplastids at frequencies approaching 100%. The highly efficient genome editing via SaCas9/sgRNA RNPs was obtained for both reporter and endogenous genes and observed in multiple parasite life cycle stages in various strains of T. cruzi, as well as in T. brucei and Leishmania major. RNP complex delivery was also used to successfully tag proteins at endogenous loci and to assess the biological functions of essential genes. Thus, the use of SaCas9 RNP complexes for gene editing in kinetoplastids provides a simple, rapid, and cloning- and selection-free method to assess gene function in these important human pathogens.

  10. Les kystes hydatiques du foie rompus dans les voies biliaires: à propos de 120 cas

    Science.gov (United States)

    Moujahid, Mountassir; Tajdine, Mohamed Tarik

    2011-01-01

    Etude rétrospective rapportant une série de kystes hydatiques rompus dans les voies biliaires colligés dans le service de chirurgie de l'hôpital militaire Avicenne à Marrakech. Entre 1990 à 2008, sur 536 kystes hydatiques du foie opérés dans le service, 120 étaient compliqués de rupture dans les voies biliaires soit 22,38%. Il y avait 82hommes et 38 femmes. L’âge moyen était de 35 ans avec des extrêmes allant de 10 à 60 ans. La clinique était dominée par la crise d'angiocholite ou une douleur du flanc droit. L'ictère était isolé dans huit cas. La fistule biliokystique était latente dans plus de 50% des cas. Le traitement a consisté en une résection du dôme saillant dans103cas (85,84%), une périkystectomie chez 11 malades (9,16%) et une lobectomie gauche dans six cas (5%). Le traitement de la fistule bilio kystique a consisté en une suture chez 36malades et un drainage bipolaire dans 25 cas, La déconnexion kysto-biliaire ou cholédocotomie trans hépatico kystique selon Perdomo était pratiquée dans 49cas et une anastomose bilio-digestive cholédoco-duodénale dans 10 cas. La durée moyenne d'hospitalisation était de 20jours. Nous déplorons deux décès par choc septique et un troisième par encéphalopathie secondaire à une cirrhose biliaire. La morbidité était représentée par huit abcès sous phrénique, douze fistules biliaires prolongées et deux occlusions intestinales. Les kystes hydatiques rompus dans les voies biliaires représentent la complication la plus grave de cette pathologie bénigne. Le traitement repose sur des méthodes radicales qui sont d'une efficacité reconnue, mais de réalisation dangereuse et les méthodes conservatrices, en particulier la déconnexion kysto-biliaire qui est une méthode simple et qui donne de bons résultats à court et à long terme. PMID:22384289

  11. CAS Introduction to Accelerator Physics in Bulgaria

    CERN Multimedia

    CERN Bulletin

    2010-01-01

    The CERN Accelerator School (CAS) and the Institute for Nuclear Research & Nuclear Energy (INRNE – Bulgarian Academy of Sciences) jointly organised a course on Introduction to Accelerators, at the Grand Hotel Varna, Bulgaria, from 19 September to 1 October, 2010.   CERN Accelerator School group photo. The course was extremely well attended with 109 participants representing 34 different nationalities, coming from countries as far away as Australia, Canada and Vietnam. The intensive programme comprised 39 lectures, 3 seminars, 4 tutorials where the students were split into three groups, a poster session where students could present their own work, and 7 hours of guided and private study. Feedback from the participants was extremely positive, praising the expertise and enthusiasm of the lecturers, as well as the high standard and excellent quality of their lectures. For the first time at CAS, the CERN Director-General, Rolf Heuer, visited the school and presented a seminar entitled...

  12. CAS - Great success for the DSP course

    CERN Multimedia

    2007-01-01

    The CERN Accelerator School (CAS) and the Uppsala University jointly organized a specialized school on "Digital Signal Processing" in Sigtuna, Sweden from 1-9 June, 2007. This course was a "première" in many ways: firstly the topic had never been addressed by CAS, and secondly the structure of the course differed from the usual specialized courses in the sense that it was composed of 32 hours of theoretical lectures in the mornings and 16 hours "hands-on" courses in the afternoons. The latter, which have been designed by CERN experts, had some logistic implications in transporting computers and circuit boards (DSP and FPGA) to Sweden. The principle of this new approach was extremely well received by the accelerator community and 97 participants representing 23 different nationalities (80% of the participants originating from the CERN Member States) attended the course. As illustrated by the very positive feedback received from th...

  13. Le mal de Pott: à propos de 82 cas

    Directory of Open Access Journals (Sweden)

    Badr Fedoul

    2011-03-01

    Full Text Available Nous rapportant dans cette étude, les résultats de l’expérience du service de neurochirurgie du CHU Hassan II de Fès dans la prise en charge du mal de pott dans la région de Fès. Il s’agit d’une étude rétrospective de quatre-vingt-deux cas; étalée sur une période de cinq ans (janvier 2002 au décembre 2006. L’objectif de ce travail était d’illustrer les différents aspects épidémiologiques, diagnostiques et thérapeutiques de la localisation vertébrale de la tuberculose dans notre pratique. L'âge moyen de nos patients était de 43,1 ans, avec une légère prédominance féminine (53,82%. La durée d'évolution de la maladie était longue (dix mois en moyenne; ceci est expliquée par la symptomatologie initiale insidieuse faite de rachialgies (98,78% et une admission des patients au stade de complications neurologiques (41,46%. La radiographie standard était réalisée chez tous nos patients, et complétée par la TDM dans 86.58% des cas ce qui a permis de déceler la prédominance de l'atteinte dorsale et lombaire. L'IRM est l'examen de choix, elle était demandée chez tous les malades déficitaires (37,8%.Tous nos patients ont bénéficié d'un traitement antibacillaire associé à une immobilisation du foyer pottique. Une décompression par voie antérieure était réalisée chez 29 patients (35,36 %; alors que la laminectomie n'était pratiquée que chez 5 patients (6.09 %, tandis que l'évacuation de l'abcès de psoas était réalisée chez 25 patients (30,48 %. Le diagnostic de certitude histologique était posé dans 51 cas (62,19%. Les meilleurs résultats étaient obtenus chez les malades opérés par voie antérieure, 26 cas (89,65% de récupération totale et 3 cas (10,34% partielle. L'évolution vers la consolidation et la fusion vertébrale était la règle chez tous nos malades et ceci au bout de 4 à 18 mois après le traitement.

  14. Annotation and Classification of CRISPR-Cas Systems.

    Science.gov (United States)

    Makarova, Kira S; Koonin, Eugene V

    2015-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) is a prokaryotic adaptive immune system that is represented in most archaea and many bacteria. Among the currently known prokaryotic defense systems, the CRISPR-Cas genomic loci show unprecedented complexity and diversity. Classification of CRISPR-Cas variants that would capture their evolutionary relationships to the maximum possible extent is essential for comparative genomic and functional characterization of this theoretically and practically important system of adaptive immunity. To this end, a multipronged approach has been developed that combines phylogenetic analysis of the conserved Cas proteins with comparison of gene repertoires and arrangements in CRISPR-Cas loci. This approach led to the current classification of CRISPR-Cas systems into three distinct types and ten subtypes for each of which signature genes have been identified. Comparative genomic analysis of the CRISPR-Cas systems in new archaeal and bacterial genomes performed over the 3 years elapsed since the development of this classification makes it clear that new types and subtypes of CRISPR-Cas need to be introduced. Moreover, this classification system captures only part of the complexity of CRISPR-Cas organization and evolution, due to the intrinsic modularity and evolutionary mobility of these immunity systems, resulting in numerous recombinant variants. Moreover, most of the cas genes evolve rapidly, complicating the family assignment for many Cas proteins and the use of family profiles for the recognition of CRISPR-Cas subtype signatures. Further progress in the comparative analysis of CRISPR-Cas systems requires integration of the most sensitive sequence comparison tools, protein structure comparison, and refined approaches for comparison of gene neighborhoods.

  15. Annotation and Classification of CRISPR-Cas Systems

    Science.gov (United States)

    Makarova, Kira S.; Koonin, Eugene V.

    2018-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) is a prokaryotic adaptive immune system that is represented in most archaea and many bacteria. Among the currently known prokaryotic defense systems, the CRISPR-Cas genomic loci show unprecedented complexity and diversity. Classification of CRISPR-Cas variants that would capture their evolutionary relationships to the maximum possible extent is essential for comparative genomic and functional characterization of this theoretically and practically important system of adaptive immunity. To this end, a multipronged approach has been developed that combines phylogenetic analysis of the conserved Cas proteins with comparison of gene repertoires and arrangements in CRISPR-Cas loci. This approach led to the current classification of CRISPR-Cas systems into three distinct types and ten subtypes for each of which signature genes have been identified. Comparative genomic analysis of the CRISPR-Cas systems in new archaeal and bacterial genomes performed over the 3 years elapsed since the development of this classification makes it clear that new types and subtypes of CRISPR-Cas need to be introduced. Moreover, this classification system captures only part of the complexity of CRISPR-Cas organization and evolution, due to the intrinsic modularity and evolutionary mobility of these immunity systems, resulting in numerous recombinant variants. Moreover, most of the cas genes evolve rapidly, complicating the family assignment for many Cas proteins and the use of family profiles for the recognition of CRISPR-Cas subtype signatures. Further progress in the comparative analysis of CRISPR-Cas systems requires integration of the most sensitive sequence comparison tools, protein structure comparison, and refined approaches for comparison of gene neighborhoods. PMID:25981466

  16. La tuberculose sternale: à propos de 2 cas | Ouarssani | Pan African ...

    African Journals Online (AJOL)

    On rapporte deux cas de tuberculose sternale âgés respectivement de 45 ans et de 10ans, colligés au service de pneumologie, l'examen histologique et bactériologique ont permis d'affirmer le diagnostic de tuberculose, et le traitement antibacillaire a permis une évolution favorable .A la lumière de ces observations, les ...

  17. Services

    International Nuclear Information System (INIS)

    Hardeman, F.

    1998-01-01

    The objectives of the services section is (1) to offer complete services in health-physics measurements according to international quality standards, (2) to improve continuously these measurement techniques and to follow up international recommendations and legislation concerning the surveillance of workers, (3) to support and advise nuclear and non-nuclear industry on problems of radioactive contamination. Achievements related to gamma spectrometry, whole-body counting, beta and alpha spectrometry, dosimetry, radon measurements, calibration, instrumentation, and neutron activation analysis are described

  18. 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....

  19. CRISPR-Cas9 Based Engineering of Actinomycetal Genomes

    DEFF Research Database (Denmark)

    Tong, Yaojun; Charusanti, Pep; Zhang, Lixin

    2015-01-01

    . To facilitate the genetic manipulation of actinomycetes, we developed a highly efficient CRISPR-Cas9 system to delete gene(s) or gene cluster(s), implement precise gene replacements, and reversibly control gene expression in actinomycetes. We demonstrate our system by targeting two genes, actIORF1 (SCO5087......) and actVB (SCO5092), from the actinorhodin biosynthetic gene cluster in Streptomyces coelicolor A3(2). Our CRISPR-Cas9 system successfully inactivated the targeted genes. When no templates for homology-directed repair (HDR) were present, the site-specific DNA double-strand breaks (DSBs) introduced by Cas9....... Moreover, we developed a system to efficiently and reversibly control expression of target genes, deemed CRISPRi, based on a catalytically dead variant of Cas9 (dCas9). The CRISPR-Cas9 based system described here comprises a powerful and broadly applicable set of tools to manipulate actinomycetal genomes....

  20. 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.

  1. 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.

  2. 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

  3. Comparative study between RDC number 20 from ANVISA of 2006 and standard CNEN NN 6.10 of 2014 on radiotherapy services

    International Nuclear Information System (INIS)

    Silva, D.R.; Geraldo, J.M.; Batista, A.S.M.

    2017-01-01

    Introduction: The internal procedures of a Radiotherapy Service are performed based on resolutions and standards of the control bodies, both health and radiation use. In the health area, the control is carried out by the National Health Surveillance Agency (ANVISA), through the Resolution of the Collegiate Board of Directors (RDC) number 20 of 2006. On the other hand, because it is a service that uses high energy ionizing radiation, it must comply with the rules of the National Nuclear Energy Commission (CNEN) specifically CNEN NN 6.10 of 2014. It is therefore necessary to integrate the recommendations contained in the ANVISA and CNEN determinations, requiring interpretation and transposition effort for the internal regime of procedures of each institution. Methods: The objective of this study was to compare, discuss and interpret RDC number 20 and CNEN NN 6.10 in relation to the understanding of how the two contribute to the applicability of radioprotection and radiation therapy rules. Results: Tables are presented in constant or not in the two documents and evaluated the contributions and focus of each of them. Conclusion: It is noted that each text is reconciled with the interests of each legislator and supervisory body, that is, CNEN and ANVISA. In the control of the use of radioactive sources and emitting equipment, in the case of CNEN, and in the control of agents harmful to health, ANVISA

  4. La tuberculose extra-ganglionnaire primitive de la sphère ORL: à propos de 15 cas

    Science.gov (United States)

    Touati, Mohamed Mliha; Darouassi, Youssef; Chihani, Mehdi; Lakouichmi, Mohammed; Tourabi, Khalid; Ammar, Haddou; Bouaity, Brahim

    2014-01-01

    Les localisations ORL extra ganglionnaires de la tuberculose sont rares. La symptomatologie clinique ainsi que les examens paracliniques sont souvent trompeurs,posant ainsi le problème de diagnostic différentiel avec la pathologie tumorale. Nous rapportons 15 cas de localisations extra ganglionnaires de tuberculose, colligés au service ORL et CCF de l'Hopital Militaire Avicenne de Marrakech colligés entre 2009 et 2013. L’âge moyen de nos patients est de 33 ans. L’étude topographique a montré 6 cas au niveau du cavum, un cas de miliaire tuberculeuse pharyngée, 4 cas laryngés; 2 localisations auriculaires; 1 parotidienne et 1 localisation sous maxillaire. Le diagnostic était anatomopathologiquedans tous les cas. Tous nos patients ont reçu un traitement antituberculeux avec une bonne évolution. Mots-clés: Tuberculose, amygdale, rhinopharynx, larynx, glandes salivaires,Oreille moyenne. PMID:25815100

  5. Does access to general dental treatment affect the number and complexity of patients presenting to the acute hospital service with severe dentofacial infections?

    Science.gov (United States)

    Bowe, Conor M; Gargan, Mary Louise; Kearns, Gerard J; Stassen, Leo F A

    2015-01-01

    This is a retrospective study to review the treatment and management of patients presenting with odontogenic infections in a large urban teaching hospital over a four-year period, comparing the number and complexity of odontogenic infections presenting to an acute general hospital in two periods, as follows: Group A (January 2008 to March 2010) versus Group B (April 2010 to December 2011). The background to the study is 'An alteration in patient access to primary dental care instituted by the Department of Health in April 2010'. a) to identify any alteration in the pattern and complexity of patients' presentation with odontogenic infections following recent changes in access to treatment via the Dental Treatment Services Scheme (DTSS) and the Dental Treatment Benefit Scheme (DTBS) in April 2010; and, b) to evaluate the management of severe odontogenic infections. Data was collated by a combination of a comprehensive chart review and electronic patient record analysis based on the primary discharge diagnosis as recorded in the Hospital In-Patient Enquiry (HIPE) system. Fifty patients were admitted to the National Maxillofacial Unit, St James's Hospital, under the oral and maxillofacial service over a four-year period, with an odontogenic infection as the primary diagnosis. There was an increased number of patients presenting with odontogenic infections during Group B of the study. These patients showed an increased complexity and severity of infection. Although there was an upward trend in the numbers and complexity of infections, this trending did not reach statistical significance. The primary cause of infection was dental caries in all patients. Dental caries is a preventable and treatable disease. Increased resources should be made available to support access to dental care, and thereby lessen the potential for the morbidity and mortality associated with serious odontogenic infections. The study at present continues as a prospective study.

  6. Expanding the CRISPR/Cas9 toolkit for Pichia pastoris with efficient donor integration and alternative resistance markers.

    Science.gov (United States)

    Weninger, Astrid; Fischer, Jasmin E; Raschmanová, Hana; Kniely, Claudia; Vogl, Thomas; Glieder, Anton

    2018-04-01

    Komagataella phaffii (syn. Pichia pastoris) is one of the most commonly used host systems for recombinant protein expression. Achieving targeted genetic modifications had been hindered by low frequencies of homologous recombination (HR). Recently, a CRISPR/Cas9 genome editing system has been implemented for P. pastoris enabling gene knockouts based on indels (insertion, deletions) via non-homologous end joining (NHEJ) at near 100% efficiency. However, specifically integrating homologous donor cassettes via HR for replacement studies had proven difficult resulting at most in ∼20% correct integration using CRISPR/Cas9. Here, we demonstrate the CRISPR/Cas9 mediated integration of markerless donor cassettes at efficiencies approaching 100% using a ku70 deletion strain. The Ku70p is involved in NHEJ repair and lack of the protein appears to favor repair via HR near exclusively. While the absolute number of transformants in the Δku70 strain is reduced, virtually all surviving transformants showed correct integration. In the wildtype strain, markerless donor cassette integration was also improved up to 25-fold by placing an autonomously replicating sequence (ARS) on the donor cassette. Alternative strategies for improving donor cassette integration using a Cas9 nickase variant or reducing off targeting associated toxicity using a high fidelity Cas9 variant were so far not successful in our hands in P. pastoris. Furthermore we provide Cas9/gRNA expression plasmids with a Geneticin resistance marker which proved to be versatile tools for marker recycling. The reported CRSIPR-Cas9 tools can be applied for modifying existing production strains and also pave the way for markerless whole genome modification studies in P. pastoris. © 2017 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.

  7. CAS - CERN Accelerator School: Power Converters

    CERN Document Server

    2015-01-01

    These proceedings collate lectures given at the twenty-eighth specialized course organised by the CERN Accelerator School (CAS). The course was held at the Hotel du Parc, Baden, Switzerland from 7 - 14 May 2014, in collaboration with the Paul Scherrer Institute. Following introductory lectures on accelerators and the requirements on power converters, the course covered components and topologies of the different types of power converters needed for particle accelerators. Issues of design, control and exploitation in a sometimes-hostile environment were addressed. Site visits to ABB and PSI provided an insight into state-of-the-art power converter production and operation, while topical seminars completed the programme.

  8. Highly specific targeted mutagenesis in plants using Staphylococcus aureus Cas9

    OpenAIRE

    Hidetaka Kaya; Masafumi Mikami; Akira Endo; Masaki Endo; Seiichi Toki

    2016-01-01

    The CRISPR/Cas9 system is an efficient and convenient tool for genome editing in plants. Cas9 nuclease derived from Streptococcus pyogenes (Sp) is commonly used in this system. Recently, Staphylococcus aureus Cas9 (SaCas9)-mediated genome editing was reported in human cells and Arabidopsis. Because SaCas9 (1053 a.a.) is smaller than SpCas9 (1368 a.a.), SaCas9 could have substantial advantages for delivering and expressing Cas9 protein, especially using virus vectors. Since the protospacer adj...

  9. A newly discovered Bordetella species carries a transcriptionally active CRISPR-Cas with a small Cas9 endonuclease.

    Science.gov (United States)

    Ivanov, Yury V; Shariat, Nikki; Register, Karen B; Linz, Bodo; Rivera, Israel; Hu, Kai; Dudley, Edward G; Harvill, Eric T

    2015-10-26

    Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) are widely distributed among bacteria. These systems provide adaptive immunity against mobile genetic elements specified by the spacer sequences stored within the CRISPR. The CRISPR-Cas system has been identified using Basic Local Alignment Search Tool (BLAST) against other sequenced and annotated genomes and confirmed via CRISPRfinder program. Using Polymerase Chain Reactions (PCR) and Sanger DNA sequencing, we discovered CRISPRs in additional bacterial isolates of the same species of Bordetella. Transcriptional activity and processing of the CRISPR have been assessed via RT-PCR. Here we describe a novel Type II-C CRISPR and its associated genes-cas1, cas2, and cas9-in several isolates of a newly discovered Bordetella species. The CRISPR-cas locus, which is absent in all other Bordetella species, has a significantly lower GC-content than the genome-wide average, suggesting acquisition of this locus via horizontal gene transfer from a currently unknown source. The CRISPR array is transcribed and processed into mature CRISPR RNAs (crRNA), some of which have homology to prophages found in closely related species B. hinzii. Expression of the CRISPR-Cas system and processing of crRNAs with perfect homology to prophages present in closely related species, but absent in that containing this CRISPR-Cas system, suggest it provides protection against phage predation. The 3,117-bp cas9 endonuclease gene from this novel CRISPR-Cas system is 990 bp smaller than that of Streptococcus pyogenes, the 4,017-bp allele currently used for genome editing, and which may make it a useful tool in various CRISPR-Cas technologies.

  10. Production of Purified CasRNPs for Efficacious Genome Editing.

    Science.gov (United States)

    Lingeman, Emily; Jeans, Chris; Corn, Jacob E

    2017-10-02

    CRISPR-Cas systems have been harnessed as modular genome editing reagents for functional genomics and show promise to cure genetic diseases. Directed by a guide RNA, a Cas effector introduces a double stranded break in DNA and host cell DNA repair leads to the introduction of errors (e.g., to knockout a gene) or a programmed change. Introduction of a Cas effector and guide RNA as a purified Cas ribonucleoprotein complex (CasRNP) has recently emerged as a powerful approach to alter cell types and organisms. Not only does CasRNP editing exhibit increased efficacy and specificity, it avoids optimization and iteration of species-specific factors such as codon usage, promoters, and terminators. CasRNP editing has been rapidly adopted for research use in many contexts and is quickly becoming a popular method to edit primary cells for therapeutic application. This article describes how to make a Cas9 RNP and outlines its use for gene editing in human cells. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  11. Engineering the Caenorhabditis elegans genome with CRISPR/Cas9

    NARCIS (Netherlands)

    Waaijers, Selma; Boxem, Mike

    2014-01-01

    The development in early 2013 of CRISPR/Cas9-based genome engineering promises to dramatically advance our ability to alter the genomes of model systems at will. A single, easily produced targeting RNA guides the Cas9 endonuclease to a specific DNA sequence where it creates a double strand break.

  12. CATO--A Guided User Interface for Different CAS

    Science.gov (United States)

    Janetzko, Hans-Dieter

    2017-01-01

    CATO is a new user interface, written in Java and developed by the author as a response to the significant difficulties faced by students who only sporadically use computer algebra systems (CAS). The usage of CAS in mathematical lectures should be an integral part of mathematical instruction. However, difficulties arise for those students who have…

  13. From Calculus to Dynamical Systems through DGS and CAS

    Science.gov (United States)

    García, Jeanett López; Zamudio, Jorge Javier Jiménez

    2015-01-01

    Several factors have motivated the use of CAS or DGS in the teaching-learning process, such as: the development of new technologies, the availability of computers, and the widespread use of the Internet, among others. Even more, the trend to include CAS and DGS in the curricula of some undergraduate studies has resulted in the instruction of the…

  14. NSP-CAS Protein Complexes: Emerging Signaling Modules in Cancer.

    Science.gov (United States)

    Wallez, Yann; Mace, Peter D; Pasquale, Elena B; Riedl, Stefan J

    2012-05-01

    The CAS (CRK-associated substrate) family of adaptor proteins comprises 4 members, which share a conserved modular domain structure that enables multiple protein-protein interactions, leading to the assembly of intracellular signaling platforms. Besides their physiological role in signal transduction downstream of a variety of cell surface receptors, CAS proteins are also critical for oncogenic transformation and cancer cell malignancy through associations with a variety of regulatory proteins and downstream effectors. Among the regulatory partners, the 3 recently identified adaptor proteins constituting the NSP (novel SH2-containing protein) family avidly bind to the conserved carboxy-terminal focal adhesion-targeting (FAT) domain of CAS proteins. NSP proteins use an anomalous nucleotide exchange factor domain that lacks catalytic activity to form NSP-CAS signaling modules. Additionally, the NSP SH2 domain can link NSP-CAS signaling assemblies to tyrosine-phosphorylated cell surface receptors. NSP proteins can potentiate CAS function by affecting key CAS attributes such as expression levels, phosphorylation state, and subcellular localization, leading to effects on cell adhesion, migration, and invasion as well as cell growth. The consequences of these activities are well exemplified by the role that members of both families play in promoting breast cancer cell invasiveness and resistance to antiestrogens. In this review, we discuss the intriguing interplay between the NSP and CAS families, with a particular focus on cancer signaling networks.

  15. Teaching Undergraduate Mathematics Using CAS Technology: Issues and Prospects

    Science.gov (United States)

    Tobin, Patrick C.; Weiss, Vida

    2016-01-01

    The use of handheld CAS technology in undergraduate mathematics courses in Australia is paradoxically shrinking under sustained disapproval or disdain from the professional mathematics community. Mathematics education specialists argue with their mathematics colleagues over a range of issues in course development and this use of CAS or even…

  16. CRISPR/Cas13 as a Tool for RNA Interference

    KAUST Repository

    Ali, Zahir

    2018-03-28

    Almost all biological processes involve RNA, making it crucial to develop tools for manipulation of the transcriptome. The bacterial CRISPR/Cas13 system was recently rewired to facilitate RNA manipulation in eukaryotes, including plants. We discuss here the opportunities and limitations of using CRISPR/Cas13 in plants for various types of RNA manipulation.

  17. Progress Scored in Management Information System at CAS

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    @@ CAS initiative to upgrade its management information system (MIS) is making significant progress. Recently, 116 CAS subordinates have completed their online trial operation of a MIS project at the Academy, called Academia Resource Planning (ARP), marking an important phased achievement of the initiative.

  18. Class 2 CRISPR-Cas RNA-guided endonucleases

    DEFF Research Database (Denmark)

    Stella, Stefano; Alcón, Pablo; Montoya, Guillermo

    2017-01-01

    CRISPR-Cas is a bacterial defense system against phage infection and nucleic acid invasion. Class 2 type II CRISPR-Cas9 has also been widely used for genome engineering. Here, we review novel insights into the CRISPR class 2 type V enzymes, specifically Cpf1 and C2c1, which display different DNA-...

  19. CRISPR/Cas9 based genome editing of Penicillium chrysogenum

    NARCIS (Netherlands)

    Pohl, Carsten; Kiel, Jan A K W; Driessen, Arnold J M; Bovenberg, Roel A L; Nygård, Yvonne

    2016-01-01

    CRISPR/Cas9 based systems have emerged as versatile platforms for precision genome editing in a wide range of organisms. Here we have developed powerful CRISPR/Cas9 tools for marker-based and marker-free genome modifications in Penicillium chrysogenum, a model filamentous fungus and industrially

  20. CAS CERN Accelerator School vacuum technology. Proceedings

    International Nuclear Information System (INIS)

    Turner, S.

    1999-01-01

    These proceedings present the lectures given at the twelfth specialized course organized by the CERN Accelerator School (CAS), the topic this time being 'Vacuum Technology'. Despite the importance of vacuum technology in the design and operation of particle accelerators at CERN and at the many other accelerators already installed around the world, this was the first time that CAS has organized a course devoted entirely to this topic. Perhaps this reflects the facts that vacuum has become one of the more critical aspects of future accelerators, and that many of the pioneers in the accelerator field are being replaced by new, younger personnel. The lectures start with the basic concepts of the physics and technology of vacuum followed by detailed descriptions of the many different types of gas-pumping devices and methods to measure the pressures achieved. The outgassing characteristics of the different materials used in the construction of vacuum systems and the optimisation of cleaning methods to reduce this outgassing are then explained together with the effects of the residual gases on the particle beams. Then follow chapters on leak detection, materials and vacuum system engineering. Finally, seminars are presented on designing vacuum systems, the history of vacuum devices, the LHC (large hadron collider) vacuum system, vacuum systems for electron storage rings, and quality assurance for vacuum. (orig.)

  1. 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.

  2. Coaching and engaging. Developing teaching with CAS in High School

    DEFF Research Database (Denmark)

    Bang, Henrik Peter; Grønbæk, Niels; Larsen, Claus Richard

    The extensive use of CAS at upper secondary school in Denmark provides a laboratory for research on the development of standards for CAS teaching. The poster focuses on action research into teachers developing lessons and student activities in an ongoing collaboration between university and high ...... schools on use of CAS in mathematics teaching. Coaches1 mediate design processes, reflection and documentation, and enable sharing. We discuss coaching as a valuable part of action research, and how to draw findings from the collaboration.......The extensive use of CAS at upper secondary school in Denmark provides a laboratory for research on the development of standards for CAS teaching. The poster focuses on action research into teachers developing lessons and student activities in an ongoing collaboration between university and high...

  3. Exploiting CRISPR-Cas to manipulate Enterococcus faecalis populations.

    Science.gov (United States)

    Hullahalli, Karthik; Rodrigues, Marinelle; Palmer, Kelli L

    2017-06-23

    CRISPR-Cas provides a barrier to horizontal gene transfer in prokaryotes. It was previously observed that functional CRISPR-Cas systems are absent from multidrug-resistant (MDR) Enterococcus faecalis , which only possess an orphan CRISPR locus, termed CRISPR2, lacking cas genes. Here, we investigate how the interplay between CRISPR-Cas genome defense and antibiotic selection for mobile genetic elements shapes in vitro E. faecalis populations. We demonstrate that CRISPR2 can be reactivated for genome defense in MDR strains. Interestingly, we observe that E. faecalis transiently maintains CRISPR targets despite active CRISPR-Cas systems. Subsequently, if selection for the CRISPR target is present, toxic CRISPR spacers are lost over time, while in the absence of selection, CRISPR targets are lost over time. We find that forced maintenance of CRISPR targets induces a fitness cost that can be exploited to alter heterogeneous E. faecalis populations.

  4. Sulfonamide inhibition studies of two β-carbonic anhydrases from the ascomycete fungus Sordaria macrospora, CAS1 and CAS2.

    Science.gov (United States)

    Vullo, Daniela; Lehneck, Ronny; Pöggeler, Stefanie; Supuran, Claudiu T

    2018-12-01

    The two β-carbonic anhydrases (CAs, EC 4.2.1.1) recently cloned and purified from the ascomycete fungus Sordaria macrospora, CAS1 and CAS2, were investigated for their inhibition with a panel of 39 aromatic, heterocyclic, and aliphatic sulfonamides and one sulfamate, many of which are clinically used agents. CAS1 was efficiently inhibited by tosylamide, 3-fluorosulfanilamide, and 3-chlorosulfanilamide (K I s in the range of 43.2-79.6 nM), whereas acetazolamide, methazolamide, topiramate, ethoxzolamide, dorzolamide, and brinzolamide were medium potency inhibitors (K I s in the range of 360-445 nM). CAS2 was less sensitive to sulfonamide inhibitors. The best CAS2 inhibitors were 5-amino-1,3,4-thiadiazole-2-sulfonamide (the deacetylated acetazolamide precursor) and 4-hydroxymethyl-benzenesulfonamide, with K I s in the range of 48.1-92.5 nM. Acetazolamide, dorzolamide, ethoxzolamide, topiramate, sulpiride, indisulam, celecoxib, and sulthiame were medium potency CAS2 inhibitors (K I s of 143-857 nM). Many other sulfonamides showed affinities in the high micromolar range or were ineffective as CAS1/2 inhibitors. Small changes in the structure of the inhibitor led to important differences of the activity. As these enzymes may show applications for the removal of anthropically generated polluting gases, finding modulators of their activity may be crucial for designing environmental-friendly CO 2 capture processes.

  5. The big bang of genome editing technology: development and application of the CRISPR/Cas9 system in disease animal models

    Science.gov (United States)

    SHAO, Ming; XU, Tian-Rui; CHEN, Ce-Shi

    2016-01-01

    Targeted genome editing technology has been widely used in biomedical studies. The CRISPR-associated RNA-guided endonuclease Cas9 has become a versatile genome editing tool. The CRISPR/Cas9 system is useful for studying gene function through efficient knock-out, knock-in or chromatin modification of the targeted gene loci in various cell types and organisms. It can be applied in a number of fields, such as genetic breeding, disease treatment and gene functional investigation. In this review, we introduce the most recent developments and applications, the challenges, and future directions of Cas9 in generating disease animal model. Derived from the CRISPR adaptive immune system of bacteria, the development trend of Cas9 will inevitably fuel the vital applications from basic research to biotechnology and biomedicine. PMID:27469250

  6. The big bang of genome editing technology: development and application of the CRISPR/Cas9 system in disease animal models.

    Science.gov (United States)

    Shao, Ming; Xu, Tian-Rui; Chen, Ce-Shi

    2016-07-18

    Targeted genome editing technology has been widely used in biomedical studies. The CRISPR-associated RNA-guided endonuclease Cas9 has become a versatile genome editing tool. The CRISPR/Cas9 system is useful for studying gene function through efficient knock-out, knock-in or chromatin modification of the targeted gene loci in various cell types and organisms. It can be applied in a number of fields, such as genetic breeding, disease treatment and gene functional investigation. In this review, we introduce the most recent developments and applications, the challenges, and future directions of Cas9 in generating disease animal model. Derived from the CRISPR adaptive immune system of bacteria, the development trend of Cas9 will inevitably fuel the vital applications from basic research to biotechnology and bio-medicine.

  7. Interference activity of a minimal Type I CRISPR–Cas system from Shewanella putrefaciens

    Science.gov (United States)

    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

  8. Interference activity of a minimal Type I CRISPR-Cas system from Shewanella putrefaciens.

    Science.gov (United States)

    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.

  9. Mr.CAS-A minimalistic (pure) Ruby CAS for fast prototyping and code generation

    Science.gov (United States)

    Ragni, Matteo

    There are Computer Algebra System (CAS) systems on the market with complete solutions for manipulation of analytical models. But exporting a model that implements specific algorithms on specific platforms, for target languages or for particular numerical library, is often a rigid procedure that requires manual post-processing. This work presents a Ruby library that exposes core CAS capabilities, i.e. simplification, substitution, evaluation, etc. The library aims at programmers that need to rapidly prototype and generate numerical code for different target languages, while keeping separated mathematical expression from the code generation rules, where best practices for numerical conditioning are implemented. The library is written in pure Ruby language and is compatible with most Ruby interpreters.

  10. Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains.

    Science.gov (United States)

    Lian, Jiazhang; Bao, Zehua; Hu, Sumeng; Zhao, Huimin

    2018-06-01

    The CRISPR/Cas9 system has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. However, its application in manipulating industrial yeast strains is less successful, probably due to the genome complexity and low copy numbers of gRNA expression plasmids. Here we developed an efficient CRISPR/Cas9 system for industrial yeast strain engineering by using our previously engineered plasmids with increased copy numbers. Four genes in both a diploid strain (Ethanol Red, 8 alleles in total) and a triploid strain (ATCC 4124, 12 alleles in total) were knocked out in a single step with 100% efficiency. This system was used to construct xylose-fermenting, lactate-producing industrial yeast strains, in which ALD6, PHO13, LEU2, and URA3 were disrupted in a single step followed by the introduction of a xylose utilization pathway and a lactate biosynthetic pathway on auxotrophic marker plasmids. The optimized CRISPR/Cas9 system provides a powerful tool for the development of industrial yeast based microbial cell factories. © 2018 Wiley Periodicals, Inc.

  11. Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9.

    Science.gov (United States)

    Ren, Xingjie; Sun, Jin; Housden, Benjamin E; Hu, Yanhui; Roesel, Charles; Lin, Shuailiang; Liu, Lu-Ping; Yang, Zhihao; Mao, Decai; Sun, Lingzhu; Wu, Qujie; Ji, Jun-Yuan; Xi, Jianzhong; Mohr, Stephanie E; Xu, Jiang; Perrimon, Norbert; Ni, Jian-Quan

    2013-11-19

    The ability to engineer genomes in a specific, systematic, and cost-effective way is critical for functional genomic studies. Recent advances using the CRISPR-associated single-guide RNA system (Cas9/sgRNA) illustrate the potential of this simple system for genome engineering in a number of organisms. Here we report an effective and inexpensive method for genome DNA editing in Drosophila melanogaster whereby plasmid DNAs encoding short sgRNAs under the control of the U6b promoter are injected into transgenic flies in which Cas9 is specifically expressed in the germ line via the nanos promoter. We evaluate the off-targets associated with the method and establish a Web-based resource, along with a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Finally, we discuss the advantages of our method in comparison with other recently published approaches.

  12. CAS Introduction to Accelerator Physics in Spain

    CERN Multimedia

    CERN Bulletin

    2012-01-01

    The CERN Accelerator School (CAS) and the University of Granada jointly organised a course called "Introduction to Accelerator Physics" in Granada, Spain, from 28 October to 9 November, 2012.   The course attracted over 200 applicants, of whom 139 were selected to attend. The students were of 25 different nationalities, coming from countries as far away as Australia, China, Guatemala and India. The intensive programme comprised 38 lectures, 3 seminars, 4 tutorials where the students were split into three groups, a poster session and 7 hours of guided and private study. Feedback from the students was very positive, praising the expertise of the lecturers, as well as the high standard and quality of their lectures. CERN's Director-General, Rolf Heuer, gave a public lecture at the Parque de las Ciencias entitled "The Large Hadron Collider: Unveiling the Universe". In addition to the academic programme, the students had the opportunity to visit the well...

  13. The ultraviolet variations of iota Cas

    Science.gov (United States)

    Molnar, M. R.; Mallama, A. D.; Soskey, D. G.; Holm, A. V.

    1976-01-01

    The Ap variable star iota Cas was observed with the photometers on OAO-2 covering the spectral range 1430-4250 A. The ultraviolet light curves show a double wave with primary minimum and maximum at phase ? 0.00 and 0.35, respectively. Secondary minimum light is at phase ? 0.65 with secondary maximum at phase ? 0.85. The light curves longward of 3150 A vary in opposition to those shortward of this 'null region'. Ground-based coude spectra show that the Fe II and Cr II line strengths have a double-wave variation such that maximum strength occurs at minimum ultraviolet light. We suggest that the strong ultraviolet opacities due to photoionization and line blanketing by these metals may cause the observed photometric variations. We have also constructed an oblique-rotator model which shows iron and chromium lying in a great circle band rather than in circular spots.

  14. CAS Accelerator Physics held in Erice, Italy

    CERN Multimedia

    CERN Accelerator School

    2013-01-01

    The CERN Accelerator School (CAS) recently organised a specialised course on Superconductivity for Accelerators, held at the Ettore Majorana Foundation and Centre for Scientific Culture in Erice, Italy from 24 April-4 May, 2013.   Photo courtesy of Alessandro Noto, Ettore Majorana Foundation and Centre for Scientific Culture. Following a handful of summary lectures on accelerator physics and the fundamental processes of superconductivity, the course covered a wide range of topics related to superconductivity and highlighted the latest developments in the field. Realistic case studies and topical seminars completed the programme. The school was very successful with 94 participants representing 23 nationalities, coming from countries as far away as Belorussia, Canada, China, India, Japan and the United States (for the first time a young Ethiopian lady, studying in Germany, attended this course). The programme comprised 35 lectures, 3 seminars and 7 hours of case study. The case studies were p...

  15. CAS Accelerator Physics (Ion Sources) in Slovakia

    CERN Multimedia

    CAS School

    2012-01-01

    The CERN Accelerator School (CAS) and the Slovak University of Technology jointly organised a specialised course on ion sources, held at the Hotel Senec, Senec, Slovakia, from 29 May to 8 June, 2012.   Following some background lectures on accelerator physics and the fundamental processes of atomic and plasma physics, the course covered a wide range of topics related to ion sources and highlighted the latest developments in the field. Realistic case studies and topical seminars completed the programme. The school was very successful, with 69 participants representing 25 nationalities. Feedback from the participants was extremely positive, reflecting the high standard of the lectures. The case studies were performed with great enthusiasm and produced some excellent results. In addition to the academic programme, the participants were able to take part in a one-day excursion consisting of a guided tour of Bratislava and free time. A welcome event was held at the Hotel Senec, with s...

  16. Adaptation in CRISPR-Cas Systems.

    Science.gov (United States)

    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.

  17. CAS medium-size nuclear plants

    International Nuclear Information System (INIS)

    Vogelweith, L.; Weiss, A.

    1977-01-01

    CEA has developed a range of pressurized water reactors of the type CAS Compact, which are adapted to civil ship propulsion, or to electric power production, combined possibly with heat production, up to outputs equivalent to 125MW(e). Nuclear plants equipped with these reactors are suitable for medium-size electric networks, especially in developing countries, because they are easily adaptable, owing to their flexibility; they can be installed and used in a variety of ways (on land, floating installation, combination of electric power and other production, etc.); they can be used as training reactors by countries wishing to limit their investment plans before undertaking a wider nuclear development. Examples of two possible realizations are presented: as a floating plant, and as a combined electric and desalting plant. (author)

  18. Nucleosome breathing and remodeling constrain CRISPR-Cas9 function

    Science.gov (United States)

    Isaac, R Stefan; Jiang, Fuguo; Doudna, Jennifer A; Lim, Wendell A; Narlikar, Geeta J; Almeida, Ricardo

    2016-01-01

    The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allow Cas9 to effectively act on chromatin in vivo. DOI: http://dx.doi.org/10.7554/eLife.13450.001 PMID:27130520

  19. CRISPR/Cas9 for cancer research and therapy.

    Science.gov (United States)

    Zhan, Tianzuo; Rindtorff, Niklas; Betge, Johannes; Ebert, Matthias P; Boutros, Michael

    2018-04-16

    CRISPR/Cas9 has become a powerful method for making changes to the genome of many organisms. First discovered in bacteria as part of an adaptive immune system, CRISPR/Cas9 and modified versions have found a widespread use to engineer genomes and to activate or to repress the expression of genes. As such, CRISPR/Cas9 promises to accelerate cancer research by providing an efficient technology to dissect mechanisms of tumorigenesis, identify targets for drug development, and possibly arm cells for cell-based therapies. Here, we review current applications of the CRISPR/Cas9 technology for cancer research and therapy. We describe novel Cas9 variants and how they are used in functional genomics to discover novel cancer-specific vulnerabilities. Furthermore, we highlight the impact of CRISPR/Cas9 in generating organoid and mouse models of cancer. Finally, we provide an overview of the first clinical trials that apply CRISPR/Cas9 as a therapeutic approach against cancer. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  20. 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.

  1. CRISPR/Cas9 Based Genome Editing of Penicillium chrysogenum.

    Science.gov (United States)

    Pohl, C; Kiel, J A K W; Driessen, A J M; Bovenberg, R A L; Nygård, Y

    2016-07-15

    CRISPR/Cas9 based systems have emerged as versatile platforms for precision genome editing in a wide range of organisms. Here we have developed powerful CRISPR/Cas9 tools for marker-based and marker-free genome modifications in Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. The developed CRISPR/Cas9 toolbox is highly flexible and allows editing of new targets with minimal cloning efforts. The Cas9 protein and the sgRNA can be either delivered during transformation, as preassembled CRISPR-Cas9 ribonucleoproteins (RNPs) or expressed from an AMA1 based plasmid within the cell. The direct delivery of the Cas9 protein with in vitro synthesized sgRNA to the cells allows for a transient method for genome engineering that may rapidly be applicable for other filamentous fungi. The expression of Cas9 from an AMA1 based vector was shown to be highly efficient for marker-free gene deletions.

  2. Engineering Plant Immunity via CRISPR/Cas13a System

    KAUST Repository

    Aljedaani, Fatimah R.

    2018-05-01

    Viral diseases constitute a major threat to the agricultural production and food security throughout the world. Plants cope with the invading viruses by triggering immune responses and small RNA interference (RNAi) systems. In prokaryotes, CRISPR/Cas systems function as an adaptive immune system to provide bacteria with resistance against invading phages and conjugative plasmids. Interestingly, CRISPR/Cas9 system was shown to interfere with eukaryotic DNA viruses and confer resistance against plant DNA viruses. The majority of the plant viruses have RNA genomes. The aim of this study is to test the ability of the newly discovered CRISPR/Cas13a immune system, that targets and cleaves single stranded RNA (ssRNA) in prokaryotes, to provide resistance against RNA viruses in plants. Here, I employ the CRISPR/Cas13a system for molecular interference against Turnip Mosaic Virus (TuMV), a plant RNA virus. The results of this study established the CRISPR/Cas13a as a molecular interference machinery against RNA viruses in plants. Specifically, my data show that the CRISPR/Cas13a machinery is able to interfere with and degrade the TuMV (TuMV-GFP) RNA genome. In conclusion, these data indicate that the CRISPR/Cas13 systems can be employed for engineering interference and durable resistance against RNA viruses in diverse plant species.

  3. CRISPR/Cas9-mediated viral interference in plants

    KAUST Repository

    Ali, Zahir

    2015-11-11

    Background The CRISPR/Cas9 system provides bacteria and archaea with molecular immunity against invading phages and conjugative plasmids. Recently, CRISPR/Cas9 has been used for targeted genome editing in diverse eukaryotic species. Results In this study, we investigate whether the CRISPR/Cas9 system could be used in plants to confer molecular immunity against DNA viruses. We deliver sgRNAs specific for coding and non-coding sequences of tomato yellow leaf curl virus (TYLCV) into Nicotiana benthamiana plants stably overexpressing the Cas9 endonuclease, and subsequently challenge these plants with TYLCV. Our data demonstrate that the CRISPR/Cas9 system targeted TYLCV for degradation and introduced mutations at the target sequences. All tested sgRNAs exhibit interference activity, but those targeting the stem-loop sequence within the TYLCV origin of replication in the intergenic region (IR) are the most effective. N. benthamiana plants expressing CRISPR/Cas9 exhibit delayed or reduced accumulation of viral DNA, abolishing or significantly attenuating symptoms of infection. Moreover, this system could simultaneously target multiple DNA viruses. Conclusions These data establish the efficacy of the CRISPR/Cas9 system for viral interference in plants, thereby extending the utility of this technology and opening the possibility of producing plants resistant to multiple viral infections.

  4. Tumeur de Frantz: deux nouveaux cas

    Science.gov (United States)

    Bellarbi, Salma; Sina, Mohamed; Jahid, Ahmed; Zouaidia, Fouad; Bernoussi, Zakia; Mahassini, Najat

    2013-01-01

    A travers cet article, nous détaillons les caractéristiques clinico-pathologiques et discutons l'histogenèse de la tumeur de Frantz. Deux patients opérés pour tumeur de Frantz. Ils ont eu un traitement chirurgical seul. L'étude morphologique était couplée à un examen immuno-histochimique (IHC) utilisant les anticorps anti CD10, anti- vimentine, anti-énolase neuronale spécifique (NSE), anti-synaptophysine, anti-chromogranine A et anti-cytokératine. Un immuno-marquage à l'anti-oestrogène et l'anti-progestérone a été réalisé dans un cas. Il s'agissait d'une femme âgée de 45ans et d'un garçon de 12 ans. Les aspects échographiques et scannographiques étaient non spécifiques. Une exérèse chirurgicale complète a été réalisée dans les deux cas. L'analyse histologique évoquait une tumeur de Frantz. Le diagnostic a été retenu après étude immuno-histohimique. L'évolution était favorable sans récidive avec respectivement un recul de 18 et 16 mois. La tumeur de Frantz est une entité rare. Son diagnostic repose sur l'examen anatomopathologique complété par l'étude immuno-histochimique. Son pronostic est excellent après résection chirurgicale. PMID:23503717

  5. Programmable RNA recognition and cleavage by CRISPR/Cas9.

    Science.gov (United States)

    O'Connell, Mitchell R; Oakes, Benjamin L; Sternberg, Samuel H; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A

    2014-12-11

    The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.

  6. 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....

  7. Optimizing CRISPR/Cas9 for the Diatom Phaeodactylum tricornutum

    Directory of Open Access Journals (Sweden)

    Daniel Stukenberg

    2018-06-01

    Full Text Available CRISPR/Cas9 is a powerful tool for genome editing. We constructed an easy-to-handle expression vector for application in the model organism Phaeodactylum tricornutum and tested its capabilities in order to apply CRISPR/Cas9 technology for our purpose. In our experiments, we targeted two different genes, screened for mutations and analyzed mutated diatoms in a three-step process. In the end, we identified cells, showing either monoallelic or homo-biallelic targeted mutations. Thus, we confirm that application of the CRISPR/Cas9 system for P. tricornutum is very promising, although, as discussed, overlooked pitfalls have to be considered.

  8. Embryonal Fyn-associated substrate (EFS) and CASS4: The lesser-known CAS protein family members.

    Science.gov (United States)

    Deneka, Alexander; Korobeynikov, Vladislav; Golemis, Erica A

    2015-10-01

    The CAS (Crk-associated substrate) adaptor protein family consists of four members: CASS1/BCAR1/p130Cas, CASS2/NEDD9/HEF1/Cas-L, CASS3/EFS/Sin and CASS4/HEPL. While CAS proteins lack enzymatic activity, they contain specific recognition and binding sites for assembly of larger signaling complexes that are essential for cell proliferation, survival, migration, and other processes. All family members are intermediates in integrin-dependent signaling pathways mediated at focal adhesions, and associate with FAK and SRC family kinases to activate downstream effectors regulating the actin cytoskeleton. Most studies of CAS proteins to date have been focused on the first two members, BCAR1 and NEDD9, with altered expression of these proteins now appreciated as influencing disease development and prognosis for cancer and other serious pathological conditions. For these family members, additional mechanisms of action have been defined in receptor tyrosine kinase (RTK) signaling, estrogen receptor signaling or cell cycle progression, involving discrete partner proteins such as SHC, NSP proteins, or AURKA. By contrast, EFS and CASS4 have been less studied, although structure-function analyses indicate they conserve many elements with the better-known family members. Intriguingly, a number of recent studies have implicated these proteins in immune system function, and the pathogenesis of developmental disorders, autoimmune disorders including Crohn's disease, Alzheimer's disease, cancer and other diseases. In this review, we summarize the current understanding of EFS and CASS4 protein function in the context of the larger CAS family group. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Centralisation, décentralisation et accès aux services urbains : le cas de l’enlèvement des ordures ménagères à Abidjan Centralization, decentralization, access to urban public services : waste collection in Abidjan

    Directory of Open Access Journals (Sweden)

    Amandine Henry

    2013-03-01

    Full Text Available Cet article traite de l’inégalité d’accès aux services publics urbains dans les pays en développement et, plus particulièrement, de la question de l’accès au service d’enlèvement des ordures ménagères à Abidjan. Cette question est étroitement liée au mouvement de centralisation, décentralisation et recentralisation qui a affecté la gestion de ce service au cours des dernières décennies. Chaque étape de l’avancée de la Côte d’Ivoire dans le processus de décentralisation a provoqué des changements institutionnels dans la gestion des ordures ménagères à Abidjan ; tous ont eu des effets spécifiques sur l’accès de la population à ce service. Ces effets sont étudiés à l’échelle des communes et des quartiers à travers une enquête de terrain, et sont liés aux caractéristiques socio-économiques des quartiers. Une analyse comparative entre deux communes de standing opposé, ainsi que l’examen des aspects historiques de la problématique, s’attachent à identifier les facteurs-clés d’une telle inégalité d’accès au service d’enlèvement des ordures ménagères à Abidjan.This paper deals with the inequality of access to the urban public utilities in developing countries, and more accurately with the issue of the access to the waste collection in Abidjan. This question is closely linked with the centralization, decentralization and recentralization movements that affected this service management for the last decades. Each stage of the decentralization process in Ivory Coast brought institutional changes in waste collection management in Abidjan. Each of them had specific consequences on the access of people to this service. These consequences are investigated on a local scale and are linked to the socio-economic characteristics of the studied areas. Together with the focus on the historical aspects of the problematic, a comparative analysis between two districts of opposite standing attempts

  10. 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.

  11. 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

  12. Engineering Plant Immunity via CRISPR/Cas13a System

    KAUST Repository

    Aljedaani, Fatimah R.

    2018-01-01

    systems function as an adaptive immune system to provide bacteria with resistance against invading phages and conjugative plasmids. Interestingly, CRISPR/Cas9 system was shown to interfere with eukaryotic DNA viruses and confer resistance against plant DNA

  13. CRISPR/Cas13 as a Tool for RNA Interference

    KAUST Repository

    Ali, Zahir; Mahas, Ahmed; Mahfouz, Magdy M.

    2018-01-01

    Almost all biological processes involve RNA, making it crucial to develop tools for manipulation of the transcriptome. The bacterial CRISPR/Cas13 system was recently rewired to facilitate RNA manipulation in eukaryotes, including plants. We discuss

  14. CRISPR/Cas9-mediated viral interference in plants

    KAUST Repository

    Ali, Zahir; Abulfaraj, Aala A.; Idris, Ali; Ali, Shawkat; Tashkandi, Manal; Mahfouz, Magdy M.

    2015-01-01

    These data establish the efficacy of the CRISPR/Cas9 system for viral interference in plants, thereby extending the utility of this technology and opening the possibility of producing plants resistant to multiple viral infections.

  15. CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity

    Science.gov (United States)

    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

  16. Two Distinct Approaches for CRISPR-Cas9-Mediated Gene Editing in Cryptococcus neoformans and Related Species.

    Science.gov (United States)

    Wang, Ping

    2018-06-27

    Cryptococcus neoformans and related species are encapsulated basidiomycetous fungi that cause meningoencephalitis in individuals with immune deficiency. This pathogen has a tractable genetic system; however, gene disruption via electroporation remains difficult, while biolistic transformation is often limited by lack of multiple genetic markers and the high initial cost of equipment. The approach using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) has become the technology of choice for gene editing in many organisms due to its simplicity, efficiency, and versatility. The technique has been successfully demonstrated in C. neoformans and Cryptococcus deneoformans in which two DNA plasmids expressing either the Streptococcus pyogenes CAS9 gene or the guide RNA (gRNA) were employed. However, potential adverse effects due to constitutive expression and the time-consuming process of constructing vectors to express each gRNA remain as a primary barrier for wide adaptation. This report describes the delivery of preassembled CRISPR-Cas9-gRNA ribonucleoproteins (RNPs) via electroporation that is able to generate edited mutant alleles. RNP-mediated CRISPR-Cas9 was used to replace the wild-type GIB2 gene encoding a Gβ-like/RACK1 Gib2 protein with a gib2 :: NAT allele via homologous recombination in both C. neoformans and C. deneoformans In addition, a DNA plasmid (pCnCas9:U6-gRNA) that expresses both Cas9 and gRNA, allowing for convenient yet low-cost DNA-mediated gene editing, is described. pCnCas9:U6-gRNA contains an endogenous U6 promoter for gRNA expression and restriction sites for one-step insertion of a gRNA. These approaches and resources provide new opportunities to accelerate genetic studies of Cryptococcus species. IMPORTANCE For genetic studies of the Cryptococcus genus, generation of mutant strains is often hampered by a limited number of selectable genetic markers, the tedious process of vector

  17. Diagnostic ?tiologique du diab?te insipide central: ? propos de 41 cas

    OpenAIRE

    Chaker, Fatma; Chihaoui, Melika; Yazidi, Meriem; Slimane, Hedia

    2016-01-01

    La survenue d'un syndrome polyuro-polydipsiqueavec des urines hypotoniques n?cessite une strat?gie diagnostique rigoureuse. Le but de cette ?tude ?tait d??tudier les modalit?s de diagnostic du diab?te insipide central. A travers une ?tude r?trospective de 41 cas de diab?te insipide central(DIC) collig?s au service d'Endocrinologie ? l'h?pital de la Rabta de Tunis, allant de l'ann?e 1990 ? l'an 2013, nous avons relev? les circonstances de d?couverte du DIC, les anomalies du bilan ant?-hypophys...

  18. Mariage non consommé et vaginisme: à propos de trois cas Clinique

    Science.gov (United States)

    2017-01-01

    Le vaginisme est un problème de couple. Il est source de mariages non consommés, d’infertilité et d’altération de la qualité de la relation sexuelle du couple. Par trois cas cliniques illustratifs rapportés de notre pratique clinique quotidienne et suivis sur deux années en consultation du service de psychiatrie de l’Hôpital Militaire Moulay Ismail de Meknès, nous essayons de clarifier les motifs de rencontre pour vaginisme, ses aspects cliniques et relationnels et ses particularités culturelles. PMID:28819482

  19. Cas4-Dependent Prespacer Processing Ensures High-Fidelity Programming of CRISPR Arrays.

    Science.gov (United States)

    Lee, Hayun; Zhou, Yi; Taylor, David W; Sashital, Dipali G

    2018-04-05

    CRISPR-Cas immune systems integrate short segments of foreign DNA as spacers into the host CRISPR locus to provide molecular memory of infection. Cas4 proteins are widespread in CRISPR-Cas systems and are thought to participate in spacer acquisition, although their exact function remains unknown. Here we show that Bacillus halodurans type I-C Cas4 is required for efficient prespacer processing prior to Cas1-Cas2-mediated integration. Cas4 interacts tightly with the Cas1 integrase, forming a heterohexameric complex containing two Cas1 dimers and two Cas4 subunits. In the presence of Cas1 and Cas2, Cas4 processes double-stranded substrates with long 3' overhangs through site-specific endonucleolytic cleavage. Cas4 recognizes PAM sequences within the prespacer and prevents integration of unprocessed prespacers, ensuring that only functional spacers will be integrated into the CRISPR array. Our results reveal the critical role of Cas4 in maintaining fidelity during CRISPR adaptation, providing a structural and mechanistic model for prespacer processing and integration. Copyright © 2018 Elsevier Inc. All rights reserved.

  20. CRISPR/Cas9: Transcending the Reality of Genome Editing.

    Science.gov (United States)

    Chira, Sergiu; Gulei, Diana; Hajitou, Amin; Zimta, Alina-Andreea; Cordelier, Pierre; Berindan-Neagoe, Ioana

    2017-06-16

    With the expansion of the microbiology field of research, a new genome editing tool arises from the biology of bacteria that holds the promise of achieving precise modifications in the genome with a simplicity and versatility that surpasses previous genome editing methods. This new technique, commonly named CRISPR/Cas9, led to a rapid expansion of the biomedical field; more specifically, cancer characterization and modeling have benefitted greatly from the genome editing capabilities of CRISPR/Cas9. In this paper, we briefly summarize recent improvements in CRISPR/Cas9 design meant to overcome the limitations that have arisen from the nuclease activity of Cas9 and the influence of this technology in cancer research. In addition, we present challenges that might impede the clinical applicability of CRISPR/Cas9 for cancer therapy and highlight future directions for designing CRISPR/Cas9 delivery systems that might prove useful for cancer therapeutics. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  1. Applications of CRISPR/Cas System to Bacterial Metabolic Engineering

    Directory of Open Access Journals (Sweden)

    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.

  2. 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).

  3. Efficient CRISPR/Cas9-based gene knockout in watermelon.

    Science.gov (United States)

    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.

  4. Mutations in Cas9 Enhance the Rate of Acquisition of Viral Spacer Sequences during the CRISPR-Cas Immune Response.

    Science.gov (United States)

    Heler, Robert; Wright, Addison V; Vucelja, Marija; Bikard, David; Doudna, Jennifer A; Marraffini, Luciano A

    2017-01-05

    CRISPR loci and their associated (Cas) proteins encode a prokaryotic immune system that protects against viruses and plasmids. Upon infection, a low fraction of cells acquire short DNA sequences from the invader. These sequences (spacers) are integrated in between the repeats of the CRISPR locus and immunize the host against the matching invader. Spacers specify the targets of the CRISPR immune response through transcription into short RNA guides that direct Cas nucleases to the invading DNA molecules. Here we performed random mutagenesis of the RNA-guided Cas9 nuclease to look for variants that provide enhanced immunity against viral infection. We identified a mutation, I473F, that increases the rate of spacer acquisition by more than two orders of magnitude. Our results highlight the role of Cas9 during CRISPR immunization and provide a useful tool to study this rare process and develop it as a biotechnological application. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Examination of CRISPR/Cas9 design tools and the effect of target site accessibility on Cas9 activity.

    Science.gov (United States)

    Lee, Ciaran M; Davis, Timothy H; Bao, Gang

    2018-04-01

    What is the topic of this review? In this review, we analyse the performance of recently described tools for CRISPR/Cas9 guide RNA design, in particular, design tools that predict CRISPR/Cas9 activity. What advances does it highlight? Recently, many tools designed to predict CRISPR/Cas9 activity have been reported. However, the majority of these tools lack experimental validation. Our analyses indicate that these tools have poor predictive power. Our preliminary results suggest that target site accessibility should be considered in order to develop better guide RNA design tools with improved predictive power. The recent adaptation of the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system for targeted genome engineering has led to its widespread application in many fields worldwide. In order to gain a better understanding of the design rules of CRISPR/Cas9 systems, several groups have carried out large library-based screens leading to some insight into sequence preferences among highly active target sites. To facilitate CRISPR/Cas9 design, these studies have spawned a plethora of guide RNA (gRNA) design tools with algorithms based solely on direct or indirect sequence features. Here, we demonstrate that the predictive power of these tools is poor, suggesting that sequence features alone cannot accurately inform the cutting efficiency of a particular CRISPR/Cas9 gRNA design. Furthermore, we demonstrate that DNA target site accessibility influences the activity of CRISPR/Cas9. With further optimization, we hypothesize that it will be possible to increase the predictive power of gRNA design tools by including both sequence and target site accessibility metrics. © 2017 The Authors. Experimental Physiology © 2017 The Physiological Society.

  6. Generalized Method for the User Evaluation of Purchased Information Services. Report Number Three; Monthly Report (October 1 to November 30, 1975).

    Science.gov (United States)

    Hall, Homer J.

    Four case histories were studied in an on-going project to develop a method for user selection of purchased scientific and technical information services. The issues involved were: (1) the value of computer search services to a small branch of a company technical library; (2) the special decision-making factors used for selecting items of very…

  7. Communication Disorders and Use of Intervention Services among Children Aged 3-17 Years: United States, 2012. NCHS Data Brief. Number 205

    Science.gov (United States)

    Black, Lindsey I.; Vahratian, Anjel; Hoffman, Howard J.

    2015-01-01

    Increasing the proportion of children with voice, swallowing, speech, or language disorders who receive intervention services is a Healthy People 2020 goal (1). Timely receipt of intervention services is shown to be effective for treatment of communication disorders (2-5). Using data from the 2012 National Health Interview Survey (NHIS), this…

  8. A Cas9 transgenic Plasmodium yoelii parasite for efficient gene editing.

    Science.gov (United States)

    Qian, Pengge; Wang, Xu; Yang, Zhenke; Li, Zhenkui; Gao, Han; Su, Xin-Zhuan; Cui, Huiting; Yuan, Jing

    2018-06-01

    The RNA-guided endonuclease Cas9 has applied as an efficient gene-editing method in malaria parasite Plasmodium. However, the size (4.2 kb) of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for genome editing in the parasites only introduced with cas9 plasmid. To establish the endogenous and constitutive expression of Cas9 protein in the rodent malaria parasite P. yoelii, we replaced the coding region of an endogenous gene sera1 with the intact SpCas9 coding sequence using the CRISPR/Cas9-mediated genome editing method, generating the cas9-knockin parasite (PyCas9ki) of the rodent malaria parasite P. yoelii. The resulted PyCas9ki parasite displays normal progression during the whole life cycle and possesses the Cas9 protein expression in asexual blood stage. By introducing the plasmid (pYCs) containing only sgRNA and homologous template elements, we successfully achieved both deletion and tagging modifications for different endogenous genes in the genome of PyCas9ki parasite. This cas9-knockin PyCas9ki parasite provides a new platform facilitating gene functions study in the rodent malaria parasite P. yoelii. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. Cas9 in Genetically Modified Food Is Unlikely to Cause Food Allergy.

    Science.gov (United States)

    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.

  10. COSMID: A Web-based Tool for Identifying and Validating CRISPR/Cas Off-target Sites

    Directory of Open Access Journals (Sweden)

    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.

  11. An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system.

    Science.gov (United States)

    Zhu, Xiaoxiao; Xu, Yajie; Yu, Shanshan; Lu, Lu; Ding, Mingqin; Cheng, Jing; Song, Guoxu; Gao, Xing; Yao, Liangming; Fan, Dongdong; Meng, Shu; Zhang, Xuewen; Hu, Shengdi; Tian, Yong

    2014-09-19

    The rapid generation of various species and strains of laboratory animals using CRISPR/Cas9 technology has dramatically accelerated the interrogation of gene function in vivo. So far, the dominant approach for genotyping of genome-modified animals has been the T7E1 endonuclease cleavage assay. Here, we present a polyacrylamide gel electrophoresis-based (PAGE) method to genotype mice harboring different types of indel mutations. We developed 6 strains of genome-modified mice using CRISPR/Cas9 system, and utilized this approach to genotype mice from F0 to F2 generation, which included single and multiplexed genome-modified mice. We also determined the maximal detection sensitivity for detecting mosaic DNA using PAGE-based assay as 0.5%. We further applied PAGE-based genotyping approach to detect CRISPR/Cas9-mediated on- and off-target effect in human 293T and induced pluripotent stem cells (iPSCs). Thus, PAGE-based genotyping approach meets the rapidly increasing demand for genotyping of the fast-growing number of genome-modified animals and human cell lines created using CRISPR/Cas9 system or other nuclease systems such as TALEN or ZFN.

  12. Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs.

    Science.gov (United States)

    Gagnon, James A; Valen, Eivind; Thyme, Summer B; Huang, Peng; Akhmetova, Laila; Ahkmetova, Laila; Pauli, Andrea; Montague, Tessa G; Zimmerman, Steven; Richter, Constance; Schier, Alexander F

    2014-01-01

    The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5' adenine were improved by rescuing 5' end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes.

  13. Comparative analysis of CRISPR-Cas systems in Klebsiella genomes.

    Science.gov (United States)

    Shen, Juntao; Lv, Li; Wang, Xudong; Xiu, Zhilong; Chen, Guoqiang

    2017-04-01

    Prokaryotic CRISPR-Cas system provides adaptive immunity against invasive genetic elements. Bacteria of the genus Klebsiella are important nosocomial opportunistic pathogens. However, information of CRISPR-Cas system in Klebsiella remains largely unknown. Here, we analyzed the CRISPR-Cas systems of 68 complete genomes of Klebsiella representing four species. All the elements for CRISPR-Cas system (cas genes, repeats, leader sequences, and PAMs) were characterized. Besides the typical Type I-E and I-F CRISPR-Cas systems, a new Subtype I system located in the ABC transport system-glyoxalase region was found. The conservation of the new subtype CRISPR system between different species showed new evidence for CRISPR horizontal transfer. CRISPR polymorphism was strongly correlated both with species and multilocus sequence types. Some results indicated the function of adaptive immunity: most spacers (112 of 124) matched to prophages and plasmids and no matching housekeeping genes; new spacer acquisition was observed within the same sequence type (ST) and same clonal complex; the identical spacers were observed only in the ancient position (far from the leader) between different STs and clonal complexes. Interestingly, a high ratio of self-targeting spacers (7.5%, 31 of 416) was found in CRISPR-bearing Klebsiella pneumoniae (61%, 11 of 18). In some strains, there even were multiple full matching self-targeting spacers. Some self-targeting spacers were conserved even between different STs. These results indicated that some unknown mechanisms existed to compromise the function of self-targets of CRISPR-Cas systems in K. pneumoniae. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. High content analysis platform for optimization of lipid mediated CRISPR-Cas9 delivery strategies in human cells.

    Science.gov (United States)

    Steyer, Benjamin; Carlson-Stevermer, Jared; Angenent-Mari, Nicolas; Khalil, Andrew; Harkness, Ty; Saha, Krishanu

    2016-04-01

    Non-viral gene-editing of human cells using the CRISPR-Cas9 system requires optimized delivery of multiple components. Both the Cas9 endonuclease and a single guide RNA, that defines the genomic target, need to be present and co-localized within the nucleus for efficient gene-editing to occur. This work describes a new high-throughput screening platform for the optimization of CRISPR-Cas9 delivery strategies. By exploiting high content image analysis and microcontact printed plates, multi-parametric gene-editing outcome data from hundreds to thousands of isolated cell populations can be screened simultaneously. Employing this platform, we systematically screened four commercially available cationic lipid transfection materials with a range of RNAs encoding the CRISPR-Cas9 system. Analysis of Cas9 expression and editing of a fluorescent mCherry reporter transgene within human embryonic kidney cells was monitored over several days after transfection. Design of experiments analysis enabled rigorous evaluation of delivery materials and RNA concentration conditions. The results of this analysis indicated that the concentration and identity of transfection material have significantly greater effect on gene-editing than ratio or total amount of RNA. Cell subpopulation analysis on microcontact printed plates, further revealed that low cell number and high Cas9 expression, 24h after CRISPR-Cas9 delivery, were strong predictors of gene-editing outcomes. These results suggest design principles for the development of materials and transfection strategies with lipid-based materials. This platform could be applied to rapidly optimize materials for gene-editing in a variety of cell/tissue types in order to advance genomic medicine, regenerative biology and drug discovery. CRISPR-Cas9 is a new gene-editing technology for "genome surgery" that is anticipated to treat genetic diseases. This technology uses multiple components of the Cas9 system to cut out disease-causing mutations

  15. Les tumeurs malignes naso-sinusiennes: à propos de 32 cas et revues de la littérature

    Science.gov (United States)

    Darouassi, Youssef; Touati, Mohamed Mliha; Chihani, Mehdi; El Alami, Jihane; Bouaity, Brahim; Ammar, Haddou

    2015-01-01

    Sous l’appellation tumeurs malignes naso-sinusiennes est regroupé un vaste éventail de tumeurs, aux histologies et localisations variées, mais aux tableaux cliniques souvent similaires. Le diagnostic de ces tumeurs est difficile, nécessitant une approche multidisciplinaire, à savoir oto-rhino-laryngologique, radiologique et anatomopathologique. Notre étude rétrospective concerne 32 cas de tumeurs malignes naso-sinusiennes, colligées au service d’ORL de l’hôpital militaire Avicenne de Marrakech, entre Janvier 2004 et Décembre 2014. L’analyse des données a noté que la fréquence des tumeurs épithéliales (75% des cas) était supérieure à celle des tumeurs non épithéliales (25% des cas), avec en tête de file l’adénocarcinome de l’ethmoïde (31,25%) et le carcinome épidermoïde du sinus maxillaire (18,75%). Ces tumeurs surviennent le plus souvent chez le sujet âgé avec une moyenne d’âge de 52 ans et une répartition équitable entre les deux sexes. Le délai de consultation moyen était de 12 mois avec une symptomatologie dominée par un syndrome nasosinusien (71,8%), associé dans certains cas à des signes ophtalmologiques (12,5%) ou neurologiques (15,6%). Tous nos patients ont bénéficié d’un examen clinique notamment endoscopique, d’une exploration radiologique des tumeurs et de leurs extensions, et d’une confirmation diagnostique par un examen anatomopathologique. Le traitement a consisté en une exérèse chirurgicale de la tumeur dès que cela était possible, soit dans 81,3% des cas (26 patients), généralement complété par un traitement adjuvant radio-chimiothérapique (77%). Le suivi à un an de nos patients a permis de noter une bonne évolution pour 08 d’entre eux (25%), une récidive dans 6 cas (18,75%), le décès de neuf patients (28,1%), et l’absence d’information concernant les autres cas (28,1%). PMID:26985260

  16. Inside Logistics - Exploring the Heart of Logistics, Volume 23, Number 3, 1999. Collocating Air Force Weapon Systems Inventory with the Defense Logistics Agency Premium Service Facility

    National Research Council Canada - National Science Library

    Murphy, Monte

    1999-01-01

    With declining defense budgets and the inherent responsibility as stewards of taxpayer dollars, the Services must continue to search for more efficient processes while ensuring the mission can be accomplished...

  17. Crunching the Numbers

    International Development Research Centre (IDRC) Digital Library (Canada)

    Operating a Demographic Surveillance System (DSS) like this one requires a blend of high-tech number-crunching ability and .... views follow a standardized format that takes several ... general levels of health and to the use of health services.

  18. Crystal Structure of the Minimal Cas9 from Campylobacter jejuni Reveals the Molecular Diversity in the CRISPR-Cas9 Systems.

    Science.gov (United States)

    Yamada, Mari; Watanabe, Yuto; Gootenberg, Jonathan S; Hirano, Hisato; Ran, F Ann; Nakane, Takanori; Ishitani, Ryuichiro; Zhang, Feng; Nishimasu, Hiroshi; Nureki, Osamu

    2017-03-16

    The RNA-guided endonuclease Cas9 generates a double-strand break at DNA target sites complementary to the guide RNA and has been harnessed for the development of a variety of new technologies, such as genome editing. Here, we report the crystal structures of Campylobacter jejuni Cas9 (CjCas9), one of the smallest Cas9 orthologs, in complex with an sgRNA and its target DNA. The structures provided insights into a minimal Cas9 scaffold and revealed the remarkable mechanistic diversity of the CRISPR-Cas9 systems. The CjCas9 guide RNA contains a triple-helix structure, which is distinct from known RNA triple helices, thereby expanding the natural repertoire of RNA triple helices. Furthermore, unlike the other Cas9 orthologs, CjCas9 contacts the nucleotide sequences in both the target and non-target DNA strands and recognizes the 5'-NNNVRYM-3' as the protospacer-adjacent motif. Collectively, these findings improve our mechanistic understanding of the CRISPR-Cas9 systems and may facilitate Cas9 engineering. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Comparison of Various Nuclear Localization Signal-Fused Cas9 Proteins and Cas9 mRNA for Genome Editing in Zebrafish.

    Science.gov (United States)

    Hu, Peinan; Zhao, Xueying; Zhang, Qinghua; Li, Weiming; Zu, Yao

    2018-03-02

    The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been proven to be an efficient and precise genome editing technology in various organisms. However, the gene editing efficiencies of Cas9 proteins with a nuclear localization signal (NLS) fused to different termini and Cas9 mRNA have not been systematically compared. Here, we compared the ability of Cas9 proteins with NLS fused to the N-, C-, or both the N- and C-termini and N-NLS-Cas9-NLS-C mRNA to target two sites in the tyr gene and two sites in the gol gene related to pigmentation in zebrafish. Phenotypic analysis revealed that all types of Cas9 led to hypopigmentation in similar proportions of injected embryos. Genome analysis by T7 Endonuclease I (T7E1) assays demonstrated that all types of Cas9 similarly induced mutagenesis in four target sites. Sequencing results further confirmed that a high frequency of indels occurred in the target sites ( tyr1 > 66%, tyr2 > 73%, gol1 > 50%, and gol2 > 35%), as well as various types (more than six) of indel mutations observed in all four types of Cas9-injected embryos. Furthermore, all types of Cas9 showed efficient targeted mutagenesis on multiplex genome editing, resulting in multiple phenotypes simultaneously. Collectively, we conclude that various NLS-fused Cas9 proteins and Cas9 mRNAs have similar genome editing efficiencies on targeting single or multiple genes, suggesting that the efficiency of CRISPR/Cas9 genome editing is highly dependent on guide RNAs (gRNAs) and gene loci. These findings may help to simplify the selection of Cas9 for gene editing using the CRISPR/Cas9 system. Copyright © 2018 Hu et al.

  20. Tuberculose pulmonaire révélée par un purpura thrombopénique chez l'enfant-à propos d'un cas clinique observé au service de pédiatrie des Cliniques Universitaires de Lubumbashi

    Science.gov (United States)

    Lubala, Toni Kasole; Mutombo, Augustin Mulangu; Munkana, Arthur Ndundula; Manika, Michel Muteya

    2012-01-01

    Nous rapportons le cas d'un enfant de 7 ans, de sexe masculin ayant présenté un purpura thrombopénique avec épistaxis, hématémèse, otorragies et pétéchies généralisées. Durant la même hospitalisation, nous avons mis en évidence une tuberculose pulmonaire documentée par la présence de bacilles acido-alcoolo résistants à l'examen des crachats. Nous avons observé une majoration du taux de plaquettes en une semaine de corticothérapie intraveineuse à haute dose, avant l'instauration d'une poly chimiothérapie antituberculeuse. Nous rappelons également la controverse que suscite la prise en charge de cette association rarement rapportée. PMID:23077696

  1. An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize.

    Science.gov (United States)

    Char, Si Nian; Neelakandan, Anjanasree K; Nahampun, Hartinio; Frame, Bronwyn; Main, Marcy; Spalding, Martin H; Becraft, Philip W; Meyers, Blake C; Walbot, Virginia; Wang, Kan; Yang, Bing

    2017-02-01

    CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease-based genome editing tools, optimization requires the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, ISU Maize CRISPR, utilizing Agrobacterium-delivered CRISPR/Cas9 for high-frequency targeted mutagenesis in maize. This system consists of an Escherichia coli cloning vector and an Agrobacterium binary vector. It can be used to clone up to four guide RNAs for single or multiplex gene targeting. We evaluated this system for its mutagenesis frequency and heritability using four maize genes in two duplicated pairs: Argonaute 18 (ZmAgo18a and ZmAgo18b) and dihydroflavonol 4-reductase or anthocyaninless genes (a1 and a4). T 0 transgenic events carrying mono- or diallelic mutations of one locus and various combinations of allelic mutations of two loci occurred at rates over 70% mutants per transgenic events in both Hi-II and B104 genotypes. Through genetic segregation, null segregants carrying only the desired mutant alleles without the CRISPR transgene could be generated in T 1 progeny. Inheritance of an active CRISPR/Cas9 transgene leads to additional target-specific mutations in subsequent generations. Duplex infection of immature embryos by mixing two individual Agrobacterium strains harbouring different Cas9/gRNA modules can be performed for improved cost efficiency. Together, the findings demonstrate that the ISU Maize CRISPR platform is an effective and robust tool to targeted mutagenesis in maize. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  2. 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.

  3. CRISPR-Cas adaptation: insights into the mechanism of action.

    Science.gov (United States)

    Amitai, Gil; Sorek, Rotem

    2016-02-01

    Since the first demonstration that CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against phages and plasmids, numerous studies have yielded key insights into the molecular mechanisms governing how these systems attack and degrade foreign DNA. However, the molecular mechanisms underlying the adaptation stage, in which new immunological memory is formed, have until recently represented a major unresolved question. In this Progress article, we discuss recent discoveries that have shown both how foreign DNA is identified by the CRISPR-Cas adaptation machinery and the molecular basis for its integration into the chromosome to form an immunological memory. Furthermore, we describe the roles of each of the specific CRISPR-Cas components that are involved in memory formation, and consider current models for their evolutionary origin.

  4. 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.

  5. 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

  6. CRISPR/Cas9 mediates efficient conditional mutagenesis in Drosophila.

    Science.gov (United States)

    Xue, Zhaoyu; Wu, Menghua; Wen, Kejia; Ren, Menda; Long, Li; Zhang, Xuedi; Gao, Guanjun

    2014-09-05

    Existing transgenic RNA interference (RNAi) methods greatly facilitate functional genome studies via controlled silencing of targeted mRNA in Drosophila. Although the RNAi approach is extremely powerful, concerns still linger about its low efficiency. Here, we developed a CRISPR/Cas9-mediated conditional mutagenesis system by combining tissue-specific expression of Cas9 driven by the Gal4/upstream activating site system with various ubiquitously expressed guide RNA transgenes to effectively inactivate gene expression in a temporally and spatially controlled manner. Furthermore, by including multiple guide RNAs in a transgenic vector to target a single gene, we achieved a high degree of gene mutagenesis in specific tissues. The CRISPR/Cas9-mediated conditional mutagenesis system provides a simple and effective tool for gene function analysis, and complements the existing RNAi approach. Copyright © 2014 Xue et al.

  7. CRISPR-Cas9 gene editing in honeybee and pig

    DEFF Research Database (Denmark)

    Pen, Anja

    2018-01-01

    Creating animal models by using genome modification has gotten significantly more accessible thanks to the CRISPR-Cas9 technique. In this study, we aimed to the implement the CRISPR-Cas9 methodology in the European honeybee (Apis mellifera) and pig (Sus scrofa) for generation of animal models. We...... want to use these animal models to study the development of honeybees and the pathology of amyotrophic lateral sclerosis (ALS) in a pig model of human disease. In order to simplify the production of these animal models, we test the use of sperm mediated gene transfer (SMGT) in combination with CRISPR...... mechanisms of honeybee development using genome modification will aid in uncovering these complex genetic regulatory systems. In honeybees, we have attempted to induce genome modification in the cinnabar gene through microinjection and feeding of CRISPR-Cas9 components to larvae. Additionally, we tested...

  8. Determination of local chromatin composition by CasID.

    Science.gov (United States)

    Schmidtmann, Elisabeth; Anton, Tobias; Rombaut, Pascaline; Herzog, Franz; Leonhardt, Heinrich

    2016-09-02

    Chromatin structure and function are determined by a plethora of proteins whose genome-wide distribution is typically assessed by immunoprecipitation (ChIP). Here, we developed a novel tool to investigate the local chromatin environment at specific DNA sequences. We combined the programmable DNA binding of dCas9 with the promiscuous biotin ligase BirA* (CasID) to biotinylate proteins in the direct vicinity of specific loci. Subsequent streptavidin-mediated precipitation and mass spectrometry identified both known and previously unknown chromatin factors associated with repetitive telomeric, major satellite and minor satellite DNA. With super-resolution microscopy, we confirmed the localization of the putative transcription factor ZNF512 at chromocenters. The versatility of CasID facilitates the systematic elucidation of functional protein complexes and locus-specific chromatin composition.

  9. 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

  10. Les Brulures Electriques par Haut Voltage - A Propos de 10 Cas

    Science.gov (United States)

    Belmir, R.; Fejjal, N.; El Omari, M.; El Mazouz, S.; Gharib, N.; Abassi, A.; Belmahi, A.

    2008-01-01

    Summary Les accidents électriques par haute tension (AEHT) provoquent des brûlures profondes par effet Joule le long des axes vasculo-nerveux entre les points d'entrée et de sortie, qui sont le siège de lésions délabrantes. Les Auteurs rapportent une série de dix cas d'AEHT admis au service de chirurgie réparatrice et de brûlés de l'Hôpital Ibn Sina de Rabat à travers laquelle ils étudient les caractéristiques épidémiologiques, cliniques et thérapeutiques. Tous les patients étaient des adultes de sexe masculin dont l'âge moyen était de 31 ans. Dans 70% des cas, ces brûlures étaient secondaires à un contact avec les distributeurs d'électricité avec une surface brûlée inférieure à 20%. Le traitement des lésions électrothermiques a nécessité des interventions itératives avec amputation des segments de membres nécrosés dans 70% des cas, dont les suites étaient marquées par des séquelles fonctionnelles invalidantes. La prévention des AEHT, en particulier pour les accidents du travail au sein des professions exposées, reste fondamentale. PMID:21991124

  11. Characterizing a thermostable Cas9 for bacterial genome editing and silencing

    NARCIS (Netherlands)

    Mougiakos, Ioannis; Mohanraju, Prarthana; Bosma, Elleke F.; Vrouwe, Valentijn; Finger Bou, Max; Naduthodi, Mihris I.S.; Gussak, Alex; Brinkman, Rudolf B.L.; Kranenburg, Van Richard; Oost, Van Der John

    2017-01-01

    CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including

  12. Characterizing a thermostable Cas9 for bacterial genome editing and silencing

    DEFF Research Database (Denmark)

    Mougiakos, Ioannis; Mohanraju, Prarthana; Bosma, Elleke Fenna

    2017-01-01

    CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including...

  13. Outsourcing Library Technical Services. A How-To-Do-It Manual for Librarians. How-To-Do-It Manuals for Librarians, Number 69.

    Science.gov (United States)

    Hirshon, Arnold; Winters, Barbara

    In the effort to reduce costs, improve productivity, enhance quality of services, and improve turnaround time for ordering, receiving, and cataloging new materials, libraries are increasingly turning to outsourcing as a strategic management tool to help them maximize use of their fiscal and human resources. This guide covers all aspects of…

  14. Chiropractic Health Care: A National Study of Cost of Education, Service Utilization, Number of Practicing Doctors of Chiropractic, and Other Key Policy Issues. Volumes I-II.

    Science.gov (United States)

    von Kuster, Thomas, Jr.

    Results from the first federally sponsored study of the chiropractic health care profession are presented, and a broad range of facts and issues of concern to policy-makers, the profession, and the public are described. The two-year project included three national surveys of: service providers (doctors of chiropractic in practice more than two…

  15. Hupa Numbers.

    Science.gov (United States)

    Bennett, Ruth, Ed.; And Others

    An introduction to the Hupa number system is provided in this workbook, one in a series of numerous materials developed to promote the use of the Hupa language. The book is written in English with Hupa terms used only for the names of numbers. The opening pages present the numbers from 1-10, giving the numeral, the Hupa word, the English word, and…

  16. Triangular Numbers

    Indian Academy of Sciences (India)

    Admin

    Triangular number, figurate num- ber, rangoli, Brahmagupta–Pell equation, Jacobi triple product identity. Figure 1. The first four triangular numbers. Left: Anuradha S Garge completed her PhD from. Pune University in 2008 under the supervision of Prof. S A Katre. Her research interests include K-theory and number theory.

  17. Proth Numbers

    Directory of Open Access Journals (Sweden)

    Schwarzweller Christoph

    2015-02-01

    Full Text Available In this article we introduce Proth numbers and prove two theorems on such numbers being prime [3]. We also give revised versions of Pocklington’s theorem and of the Legendre symbol. Finally, we prove Pepin’s theorem and that the fifth Fermat number is not prime.

  18. Disruption of FGF5 in Cashmere Goats Using CRISPR/Cas9 Results in More Secondary Hair Follicles and Longer Fibers

    Science.gov (United States)

    Zhu, Haijing; Niu, Yiyuan; Ma, Baohua; Yu, Honghao; Lei, Anmin; Yan, Hailong; Shen, Qiaoyan; Shi, Lei; Zhao, Xiaoe; Hua, Jinlian; Huang, Xingxu; Qu, Lei; Chen, Yulin

    2016-01-01

    Precision genetic engineering accelerates the genetic improvement of livestock for agriculture and biomedicine. We have recently reported our success in producing gene-modified goats using the CRISPR/Cas9 system through microinjection of Cas9 mRNA and sgRNAs targeting the MSTN and FGF5 genes in goat embryos. By investigating the influence of gene modification on the phenotypes of Cas9-mediated goats, we herein demonstrate that the utility of this approach involving the disruption of FGF5 results in increased number of second hair follicles and enhanced fiber length in Cas9-mediated goats, suggesting more cashmere will be produced. The effects of genome modifications were characterized using H&E and immunohistochemistry staining, quantitative PCR, and western blotting techniques. These results indicated that the gene modifications induced by the disruption of FGF5 had occurred at the morphological and genetic levels. We further show that the knockout alleles were likely capable of germline transmission, which is essential for goat population expansion. These results provide sufficient evidences of the merit of using the CRISPR/Cas9 approach for the generation of gene-modified goats displaying the corresponding mutant phenotypes. PMID:27755602

  19. Sagan numbers

    OpenAIRE

    Mendonça, J. Ricardo G.

    2012-01-01

    We define a new class of numbers based on the first occurrence of certain patterns of zeros and ones in the expansion of irracional numbers in a given basis and call them Sagan numbers, since they were first mentioned, in a special case, by the North-american astronomer Carl E. Sagan in his science-fiction novel "Contact." Sagan numbers hold connections with a wealth of mathematical ideas. We describe some properties of the newly defined numbers and indicate directions for further amusement.

  20. The Investigation of Structural Members Under Combined Axial and Transverse Loads. Section 1. Air Service Information Circular, Volume 5, Number 493

    National Research Council Canada - National Science Library

    Newell, J

    1924-01-01

    .... The purpose of the investigation was to make a comparative study of a number of approximate methods for the design of members under combined loads to ascertain their relative degrees of conservatism...

  1. CRISPR-Cas9-Mediated Genome Editing in Leishmania donovani.

    Science.gov (United States)

    Zhang, Wen-Wei; Matlashewski, Greg

    2015-07-21

    The prokaryotic CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, an RNA-guided endonuclease, has been shown to mediate efficient genome editing in a wide variety of organisms. In the present study, the CRISPR-Cas9 system has been adapted to Leishmania donovani, a protozoan parasite that causes fatal human visceral leishmaniasis. We introduced the Cas9 nuclease into L. donovani and generated guide RNA (gRNA) expression vectors by using the L. donovani rRNA promoter and the hepatitis delta virus (HDV) ribozyme. It is demonstrated within that L. donovani mainly used homology-directed repair (HDR) and microhomology-mediated end joining (MMEJ) to repair the Cas9 nuclease-created double-strand DNA break (DSB). The nonhomologous end-joining (NHEJ) pathway appears to be absent in L. donovani. With this CRISPR-Cas9 system, it was possible to generate knockouts without selection by insertion of an oligonucleotide donor with stop codons and 25-nucleotide homology arms into the Cas9 cleavage site. Likewise, we disrupted and precisely tagged endogenous genes by inserting a bleomycin drug selection marker and GFP gene into the Cas9 cleavage site. With the use of Hammerhead and HDV ribozymes, a double-gRNA expression vector that further improved gene-targeting efficiency was developed, and it was used to make precise deletion of the 3-kb miltefosine transporter gene (LdMT). In addition, this study identified a novel single point mutation caused by CRISPR-Cas9 in LdMT (M381T) that led to miltefosine resistance, a concern for the only available oral antileishmanial drug. Together, these results demonstrate that the CRISPR-Cas9 system represents an effective genome engineering tool for L. donovani. Leishmania donovani is the causative agent of fatal visceral leishmaniasis. To understand Leishmania infection and pathogenesis and identify new drug targets for control of leishmaniasis, more-efficient ways to manipulate this parasite genome are required. In this

  2. Les synovites villonodulaires du genou: à propos de 20 cas

    Science.gov (United States)

    Margad, Omar; Boukhris, Jalal; Azriouil, Ouahb; Daoudi, Mohamed; Mortaji, Aziz; Koulali, Khalid

    2017-01-01

    La synovite villonodulaire pigmentée (SVNP) est une prolifération bénigne rare de la synoviale des articulations, des bourses séreuses et des gaines tendineuses, d'étiopathogénie inconnue. Notre travail porte sur 20 cas de SVN du genou colligés à l'hôpital militaire Avicenne de Marrakech sur une période de 9 ans allant de janvier 2000 au décembre 2009 Il vise à identifier les spécificités de cette lésion, et à étudier ses principaux aspects anatomocliniques et pronostiques. L'incidence annuelle était de 2,2 cas par an. Ils étaient 15 hommes et 5 femmes, d'âge moyen de 32,5 ans, atteints du coté droit dans 55%des cas sous un mode mono articulaire chez 18 patients et bi articulaire chez un seul. La douleur et la tuméfaction étaient présentes dans 80% des cas, une masse palpable dans un cas, un syndrome méniscal a été retenu dans un cas, une mono arthrite septique dans 3 circonstances de même qu'un kyste poplité dans 2 autres. L'atteinte était diffuse dans 14 cas (70%), localisée dans 6 cas. L'imagerie par résonnance magnétique(IRM) pratiquée chez 5 patients était évocatrice chez 3, l'arthroscopie diagnostique a été utilisée chez 2 malades. La confirmation s'est faite à chaque fois à l'examen anatomopathlogique. Le traitement a consisté en une synovectomie subtotale dans 15 cas et en l'exérèse de la tumeur dans les autres formes localisées, 2 cas présentant une destruction ostéocartilagineuse ont nécessité une arthroplastie. L'évolution a été marquée par la survenue de 2 récidives sous la forme diffuse avec un recul de 3, 7 ans. On a noté une raideur avec atrophie quadricipitale chez 3 patients et une arthrolyse a été réalisée. Un cas de SVN confirmé par l'histologie s'est révélé être 5 mois après la synovectomie totale un Synovialosarcome monophasique envahissant l'os d'où l'indication de l'amputation. PMID:29255556

  3. Mismatch Negativity Responses in Children with a Diagnosis of Childhood Apraxia of Speech (CAS)

    Science.gov (United States)

    Froud, Karen; Khamis-Dakwar, Reem

    2012-01-01

    Purpose: To evaluate whether a hypothesis suggesting that apraxia of speech results from phonological overspecification could be relevant for childhood apraxia of speech (CAS). Method: High-density EEG was recorded from 5 children with CAS and 5 matched controls, ages 5-8 years, with and without CAS, as they listened to randomized sequences of CV…

  4. Suppression of HBV replication by the expression of nickase- and nuclease dead-Cas9.

    Science.gov (United States)

    Kurihara, Takeshi; Fukuhara, Takasuke; Ono, Chikako; Yamamoto, Satomi; Uemura, Kentaro; Okamoto, Toru; Sugiyama, Masaya; Motooka, Daisuke; Nakamura, Shota; Ikawa, Masato; Mizokami, Masashi; Maehara, Yoshihiko; Matsuura, Yoshiharu

    2017-07-21

    Complete removal of hepatitis B virus (HBV) DNA from nuclei is difficult by the current therapies. Recent reports have shown that a novel genome-editing tool using Cas9 with a single-guide RNA (sgRNA) system can cleave the HBV genome in vitro and in vivo. However, induction of a double-strand break (DSB) on the targeted genome by Cas9 risks undesirable off-target cleavage on the host genome. Nickase-Cas9 cleaves a single strand of DNA, and thereby two sgRNAs are required for inducing DSBs. To avoid Cas9-induced off-target mutagenesis, we examined the effects of the expressions of nickase-Cas9 and nuclease dead Cas9 (d-Cas9) with sgRNAs on HBV replication. The expression of nickase-Cas9 with a pair of sgRNAs cleaved the target HBV genome and suppressed the viral-protein expression and HBV replication in vitro. Moreover, nickase-Cas9 with the sgRNA pair cleaved the targeted HBV genome in mouse liver. Interestingly, d-Cas9 expression with the sgRNAs also suppressed HBV replication in vitro without cleaving the HBV genome. These results suggest the possible use of nickase-Cas9 and d-Cas9 with a pair of sgRNAs for eliminating HBV DNA from the livers of chronic hepatitis B patients with low risk of undesirable off-target mutation on the host genome.

  5. The role of Cas8 in type I CRISPR interference.

    Science.gov (United States)

    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.

  6. 41 CFR 102-33.130 - If we hire CAS, what are our management responsibilities?

    Science.gov (United States)

    2010-07-01

    ... 41 Public Contracts and Property Management 3 2010-07-01 2010-07-01 false If we hire CAS, what are our management responsibilities? 102-33.130 Section 102-33.130 Public Contracts and Property... § 102-33.130 If we hire CAS, what are our management responsibilities? If you hire CAS, you are...

  7. 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

  8. 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

  9. Inconclusive studies on possible CRISPR-Cas off-targets should ...

    Indian Academy of Sciences (India)

    Sandeep Chakraborty

    Published online: 30 April 2018. Keywords. ... in gene-editing technologies have resulted from the simplicity of the single effector (Cas9) class 2 CRISPR-Cas ... a Cas9/gRNA concentration dependence on off-target activity (Pattanayak et al.

  10. Apport de l'imagerie dans le diagnostic des sacroiliites infectieuses : à propos de 19 cas

    Science.gov (United States)

    Abid, Hanen; Chaabouni, Salim; Frikha, Faten; Toumi, Nozha; Souissi, Basma; Lahiani, Dorra; Bahloul, Zouhir; Ben Mahfoudh, Khaireddine

    2014-01-01

    Les sacro-iliites infectieuses méritent d’être mieux connues. Leur diagnostic est souvent retardé en raison d'une symptomatologie trompeuse et des diffcultés d'exploration de l'articulation sacro-iliaque. Notre travail est basé sur une étude rétrospective portant sur les cas de SII, recueillis sur une période comprise entre 1997 et 2008 dans notre centre universitaire Sfax-Tunisie. Le diagnostic de sacro-iliite était retenu en présence d'arguments cliniques et radiologiques d'atteinte sacroiliaque. Nous rapportons dix neuf cas de sacroiliites infectieuses (10 hommes et 9 femmes), avec un âge moyen de 32 ans. L'atteinte était unilatérale dans tous les cas. Les radiographies standard faites dans tous les cas ont été suggestives dans 14 cas et normales dans les autres cas. La TDM faite dans 13 cas a montré, un abcès des parties molles dans 8 cas et un séquestre osseux dans 2 cas. L'IRM réalisée dans 8 cas, a objectivé une infiltration des parties molles dans tous les cas et un abcès dans 3 cas. Le germe a été identifié dans 9 cas (3 cas de tuberculose, 3 cas de brucellose, 2 sacro-iliites à pyogène et un cas de candidose). Cette identification était faite par biopsie dans 3 cas, hémocultures dans 2 cas, prélèvement au niveau de la porte d'entrée dans 1 cas et sérodiagnostic dans 3 cas. Pour les autres cas, l'origine pyogène a été retenue sur des arguments cliniques et biologiques. L'imagerie joue un rôle primordial dans le diagnostic précoce et l'orientation étiologique d'une sacroiliite infectieuse. PMID:25120884

  11. Eulerian numbers

    CERN Document Server

    Petersen, T Kyle

    2015-01-01

    This text presents the Eulerian numbers in the context of modern enumerative, algebraic, and geometric combinatorics. The book first studies Eulerian numbers from a purely combinatorial point of view, then embarks on a tour of how these numbers arise in the study of hyperplane arrangements, polytopes, and simplicial complexes. Some topics include a thorough discussion of gamma-nonnegativity and real-rootedness for Eulerian polynomials, as well as the weak order and the shard intersection order of the symmetric group. The book also includes a parallel story of Catalan combinatorics, wherein the Eulerian numbers are replaced with Narayana numbers. Again there is a progression from combinatorics to geometry, including discussion of the associahedron and the lattice of noncrossing partitions. The final chapters discuss how both the Eulerian and Narayana numbers have analogues in any finite Coxeter group, with many of the same enumerative and geometric properties. There are four supplemental chapters throughout, ...

  12. CRISPR/Cas9 Promotes Functional Study of Testis Specific X-Linked Gene In Vivo.

    Directory of Open Access Journals (Sweden)

    Minyan Li

    Full Text Available Mammalian spermatogenesis is a highly regulated multistage process of sperm generation. It is hard to uncover the real function of a testis specific gene in vitro since the in vitro model is not yet mature. With the development of the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9 system, we can now rapidly generate knockout mouse models of testis specific genes to study the process of spermatogenesis in vivo. SYCP3-like X-linked 2 (SLX2 is a germ cell specific component, which contains a Cor1 domain and belongs to the XLR (X-linked, lymphocyte regulated family. Previous studies suggested that SLX2 might play an important role in mouse spermatogenesis based on its subcellular localization and interacting proteins. However, the function of SLX2 in vivo is still elusive. Here, to investigate the functions of SLX2 in spermatogenesis, we disrupted the Slx2 gene by using the CRISPR/Cas9 system. Since Slx2 is a testis specific X-linked gene, we obtained knockout male mice in the first generation and accelerated the study process. Compared with wild-type mice, Slx2 knockout mice have normal testis and epididymis. Histological observation of testes sections showed that Slx2 knockout affected none of the three main stages of spermatogenesis: mitosis, meiosis and spermiogenesis. In addition, we further confirmed that disruption of Slx2 did not affect the number of spermatogonial stem cells, meiosis progression or XY body formation by immunofluorescence analysis. As spermatogenesis was normal in Slx2 knockout mice, these mice were fertile. Taken together, we showed that Slx2 itself is not an essential gene for mouse spermatogenesis and CRISPR/Cas9 technique could speed up the functional study of testis specific X-linked gene in vivo.

  13. CRISPR/Cas9-mediated Dax1 knockout in the monkey recapitulates human AHC-HH.

    Science.gov (United States)

    Kang, Yu; Zheng, Bo; Shen, Bin; Chen, Yongchang; Wang, Lei; Wang, Jianying; Niu, Yuyu; Cui, Yiqiang; Zhou, Jiankui; Wang, Hong; Guo, Xuejiang; Hu, Bian; Zhou, Qi; Sha, Jiahao; Ji, Weizhi; Huang, Xingxu

    2015-12-20

    Mutations in the DAX1 locus cause X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH), which manifest with primary adrenal insufficiency and incomplete or absent sexual maturation, respectively. The associated defects in spermatogenesis can range from spermatogenic arrest to Sertoli cell only syndrome. Conclusions from Dax1 knockout mouse models provide only limited insight into AHC/HH disease mechanisms, because mouse models exhibit more extensive abnormalities in testicular development, including disorganized and incompletely formed testis cords with decreased number of peritubular myoid cells and male-to-female sex reversal. We previously reported successful clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome targeting in cynomolgus monkeys. Here, we describe a male fetal monkey in which targeted genome editing using CRISPR/Cas9 produced Dax1-null mutations in most somatic tissues and in the gonads. This DAX1-deficient monkey displayed defects in adrenal gland development and abnormal testis architecture with small cords, expanded blood vessels and extensive fibrosis. Sertoli cell formation was not affected. This phenotype strongly resembles findings in human patients with AHC-HH caused by mutations in DAX1. We further detected upregulation of Wnt/β-catenin-VEGF signaling in the fetal Dax1-deficient testis, suggesting abnormal activation of signaling pathways in the absence of DAX1 as one mechanism of AHC-HH. Our study reveals novel insight into the role of DAX1 in HH and provides proof-of-principle for the generation of monkey models of human disease via CRISPR/Cas9-mediated gene targeting. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  14. Genome Editing in Penicillium chrysogenum Using Cas9 Ribonucleoprotein Particles

    NARCIS (Netherlands)

    Pohl, Carsten; Mózsik, László; Driessen, Arnold J M; Bovenberg, Roel A L; Nygård, Yvonne I; Braman, Jeffrey Carl

    Several CRISPR/Cas9 tools have been recently established for precise genome editing in a wide range of filamentous fungi. This genome editing platform offers high flexibility in target selection and the possibility of introducing genetic deletions without the introduction of transgenic sequences .

  15. CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity.

    Science.gov (United States)

    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.

  16. Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity

    Science.gov (United States)

    Tycko, Josh; Myer, Vic E.; Hsu, Patrick D.

    2016-01-01

    Summary Advances in the development of delivery, repair, and specificity strategies for the CRISPR-Cas9 genome engineering toolbox are helping researchers understand gene function with unprecedented precision and sensitivity. CRISPR-Cas9 also holds enormous therapeutic potential for the treatment of genetic disorders by directly correcting disease-causing mutations. Although the Cas9 protein has been shown to bind and cleave DNA at off-target sites, the field of Cas9 specificity is rapidly progressing with marked improvements in guide RNA selection, protein and guide engineering, novel enzymes, and off-target detection methods. We review important challenges and breakthroughs in the field as a comprehensive practical guide to interested users of genome editing technologies, highlighting key tools and strategies for optimizing specificity. The genome editing community should now strive to standardize such methods for measuring and reporting off-target activity, while keeping in mind that the goal for specificity should be continued improvement and vigilance. PMID:27494557

  17. 48 CFR 9903.201-2 - Types of CAS coverage.

    Science.gov (United States)

    2010-10-01

    ..., OFFICE OF FEDERAL PROCUREMENT POLICY, OFFICE OF MANAGEMENT AND BUDGET PROCUREMENT PRACTICES AND COST... to the same cost accounting practices. The term includes Government-owned contractor-operated (GOCO... would prime contracts awarded to the same business unit. In measuring total net CAS-covered awards for a...

  18. Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

    Science.gov (United States)

    Burger, Alexa; Lindsay, Helen; Felker, Anastasia; Hess, Christopher; Anders, Carolin; Chiavacci, Elena; Zaugg, Jonas; Weber, Lukas M; Catena, Raul; Jinek, Martin; Robinson, Mark D; Mosimann, Christian

    2016-06-01

    CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo. © 2016. Published by The Company of Biologists Ltd.

  19. Cas A-A Y2K status report

    Science.gov (United States)

    Rudnick, L.

    2001-05-01

    Cas A has continued to be a rich source of information as new telescopes and detailed observations have become available. In this Y2K update, I focus on the observational findings over the last five years in the areas of ejecta mapping, hydrodynamic structure and kinematics, and high energy particles. Readers are unfortunately left to their own devices on the intriguing point source. .

  20. Application of the nanobiotechnology with the system CRISP-Cas

    Directory of Open Access Journals (Sweden)

    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.

  1. Phage Genetic Engineering Using CRISPR–Cas Systems

    Directory of Open Access Journals (Sweden)

    Asma Hatoum-Aslan

    2018-06-01

    Full Text Available Since their discovery over a decade ago, the class of prokaryotic immune systems known as CRISPR–Cas have afforded a suite of genetic tools that have revolutionized research in model organisms spanning all domains of life. CRISPR-mediated tools have also emerged for the natural targets of CRISPR–Cas immunity, the viruses that specifically infect bacteria, or phages. Despite their status as the most abundant biological entities on the planet, the majority of phage genes have unassigned functions. This reality underscores the need for robust genetic tools to study them. Recent reports have demonstrated that CRISPR–Cas systems, specifically the three major types (I, II, and III, can be harnessed to genetically engineer phages that infect diverse hosts. Here, the mechanisms of each of these systems, specific strategies used, and phage editing efficacies will be reviewed. Due to the relatively wide distribution of CRISPR–Cas systems across bacteria and archaea, it is anticipated that these immune systems will provide generally applicable tools that will advance the mechanistic understanding of prokaryotic viruses and accelerate the development of novel technologies based on these ubiquitous organisms.

  2. CRISPR/CAS9 based engineering of actinomycetal genomes

    DEFF Research Database (Denmark)

    2016-01-01

    The present invention relates to CRISPR/Cas-based methods for generating random-sized deletions around at least one target nucleic acid sequence, or for generating precise indels around at least one target nucleic acid sequence, or for modulating transcription of at least one target nucleic acid...

  3. 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

  4. Research Needed on the Use of CAS Standards and Guidelines.

    Science.gov (United States)

    Creamer, Don G.

    2003-01-01

    This article suggests research projects that would extend the knowledge base about the use of Council for the Advancement of Standards in Higher Education (CAS) standards and guidelines in useful ways. Included are five research questions and specific research methodologies to guide researchers. (Contains 20 references.) (Author)

  5. Preparing Students to Take SOA/CAS Exam FM/2

    Science.gov (United States)

    Marchand, Richard J.

    2014-01-01

    This paper provides suggestions for preparing students to take the actuarial examination on financial mathematics, SOA/CAS Exam FM/2. It is based on current practices employed at Slippery Rock University, a small public liberal arts university. Detailed descriptions of our Theory of Interest course and subsequent Exam FM/2 prep course are provided…

  6. GeoGebra 3D from the perspectives of elementary pre-service mathematics teachers who are familiar with a number of software programs

    Directory of Open Access Journals (Sweden)

    Serdal Baltaci

    2015-01-01

    Full Text Available Each new version of the GeoGebra dynamic mathematics software goes through updates and innovations. One of these innovations is the GeoGebra 5.0 version. This version aims to facilitate 3D instruction by offering opportunities for students to analyze 3D objects. While scanning the previous studies of GeoGebra 3D, it is seen that they mainly focus on the visualization of a problem in daily life and the dimensions of the evaluation of the process of problem solving with various variables. Therefore, this research problem was determined to reveal the opinions of pre-service elementary mathematics teachers who can use multiple software programs very well, about the usability of GeoGebra 3D. Compared to other studies conducted in this field, this study is thought to add a new dimension to the literature on GeoGebra 3D because the participants in the study had received training in using the Derive, Cabri, Cabri 3D, GeoGebra and GeoGebra 3D programs and had developed activities throughout their undergraduate programs and in some cases they were held responsible for those programs in their exams. In this research, we used the method of case study. The participants consisted of five elementary pre-service mathematics teachers who were enrolled in fourth year courses. We employed semi-structured interviews to collect data. It is concluded that pre-service elementary mathematics teachers expressed a great deal of opinions about the positive contribution of the GeoGebra 3D dynamic mathematics software.

  7. Transfinite Numbers

    Indian Academy of Sciences (India)

    Transfinite Numbers. What is Infinity? S M Srivastava. In a series of revolutionary articles written during the last quarter of the nineteenth century, the great Ger- man mathematician Georg Cantor removed the age-old mistrust of infinity and created an exceptionally beau- tiful and useful theory of transfinite numbers. This is.

  8. PinAPL-Py: A comprehensive web-application for the analysis of CRISPR/Cas9 screens.

    Science.gov (United States)

    Spahn, Philipp N; Bath, Tyler; Weiss, Ryan J; Kim, Jihoon; Esko, Jeffrey D; Lewis, Nathan E; Harismendy, Olivier

    2017-11-20

    Large-scale genetic screens using CRISPR/Cas9 technology have emerged as a major tool for functional genomics. With its increased popularity, experimental biologists frequently acquire large sequencing datasets for which they often do not have an easy analysis option. While a few bioinformatic tools have been developed for this purpose, their utility is still hindered either due to limited functionality or the requirement of bioinformatic expertise. To make sequencing data analysis of CRISPR/Cas9 screens more accessible to a wide range of scientists, we developed a Platform-independent Analysis of Pooled Screens using Python (PinAPL-Py), which is operated as an intuitive web-service. PinAPL-Py implements state-of-the-art tools and statistical models, assembled in a comprehensive workflow covering sequence quality control, automated sgRNA sequence extraction, alignment, sgRNA enrichment/depletion analysis and gene ranking. The workflow is set up to use a variety of popular sgRNA libraries as well as custom libraries that can be easily uploaded. Various analysis options are offered, suitable to analyze a large variety of CRISPR/Cas9 screening experiments. Analysis output includes ranked lists of sgRNAs and genes, and publication-ready plots. PinAPL-Py helps to advance genome-wide screening efforts by combining comprehensive functionality with user-friendly implementation. PinAPL-Py is freely accessible at http://pinapl-py.ucsd.edu with instructions and test datasets.

  9. A non-inheritable maternal Cas9-based multiple-gene editing system in mice

    OpenAIRE

    Takayuki Sakurai; Akiko Kamiyoshi; Hisaka Kawate; Chie Mori; Satoshi Watanabe; Megumu Tanaka; Ryuichi Uetake; Masahiro Sato; Takayuki Shindo

    2016-01-01

    The CRISPR/Cas9 system is capable of editing multiple genes through one-step zygote injection. The preexisting method is largely based on the co-injection of Cas9 DNA (or mRNA) and guide RNAs (gRNAs); however, it is unclear how many genes can be simultaneously edited by this method, and a reliable means to generate transgenic (Tg) animals with multiple gene editing has yet to be developed. Here, we employed non-inheritable maternal Cas9 (maCas9) protein derived from Tg mice with systemic Cas9...

  10. 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.

  11. All-in-One CRISPR-Cas9/FokI-dCas9 Vector-Mediated Multiplex Genome Engineering in Cultured Cells.

    Science.gov (United States)

    Sakuma, Tetsushi; Sakamoto, Takuya; Yamamoto, Takashi

    2017-01-01

    CRISPR-Cas9 enables highly convenient multiplex genome engineering in cultured cells, because it utilizes generic Cas9 nuclease and an easily customizable single-guide RNA (sgRNA) for site-specific DNA double-strand break induction. We previously established a multiplex CRISPR-Cas9 assembly system for constructing an all-in-one vector simultaneously expressing multiple sgRNAs and Cas9 nuclease or other Cas9 variants including FokI-dCas9, which supersedes the wild-type Cas9 with regard to high specificity. In this chapter, we describe a streamlined protocol to design and construct multiplex CRISPR-Cas9 or FokI-dCas9 vectors, to introduce them into cultured cells by lipofection or electroporation, to enrich the genomically edited cells with a transient puromycin selection, to validate the mutation efficiency by Surveyor nuclease assay, and to perform off-target analyses. We show that our protocol enables highly efficient multiplex genome engineering even in hard-to-transfect HepG2 cells.

  12. CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Jakociunas, Tadas; Rajkumar, Arun Stephen; Zhang, Jie

    2015-01-01

    , we present a method for marker-free multiloci integration of in vivo assembled DNA parts. By the use of CRISPR/Cas9-mediated one-step double-strand breaks at single, double and triple integration sites we report the successful in vivo assembly and chromosomal integration of DNA parts. We call our...

  13. Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems.

    Science.gov (United States)

    Gomaa, Ahmed A; Klumpe, Heidi E; Luo, Michelle L; Selle, Kurt; Barrangou, Rodolphe; Beisel, Chase L

    2014-01-28

    CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems in bacteria and archaea employ CRISPR RNAs to specifically recognize the complementary DNA of foreign invaders, leading to sequence-specific cleavage or degradation of the target DNA. Recent work has shown that the accidental or intentional targeting of the bacterial genome is cytotoxic and can lead to cell death. Here, we have demonstrated that genome targeting with CRISPR-Cas systems can be employed for the sequence-specific and titratable removal of individual bacterial strains and species. Using the type I-E CRISPR-Cas system in Escherichia coli as a model, we found that this effect could be elicited using native or imported systems and was similarly potent regardless of the genomic location, strand, or transcriptional activity of the target sequence. Furthermore, the specificity of targeting with CRISPR RNAs could readily distinguish between even highly similar strains in pure or mixed cultures. Finally, varying the collection of delivered CRISPR RNAs could quantitatively control the relative number of individual strains within a mixed culture. Critically, the observed selectivity and programmability of bacterial removal would be virtually impossible with traditional antibiotics, bacteriophages, selectable markers, or tailored growth conditions. Once delivery challenges are addressed, we envision that this approach could offer a novel means to quantitatively control the composition of environmental and industrial microbial consortia and may open new avenues for the development of "smart" antibiotics that circumvent multidrug resistance and differentiate between pathogenic and beneficial microorganisms. Controlling the composition of microbial populations is a critical aspect in medicine, biotechnology, and environmental cycles. While different antimicrobial strategies, such as antibiotics, antimicrobial peptides, and lytic bacteriophages, offer partial solutions

  14. Chocolate Numbers

    OpenAIRE

    Ji, Caleb; Khovanova, Tanya; Park, Robin; Song, Angela

    2015-01-01

    In this paper, we consider a game played on a rectangular $m \\times n$ gridded chocolate bar. Each move, a player breaks the bar along a grid line. Each move after that consists of taking any piece of chocolate and breaking it again along existing grid lines, until just $mn$ individual squares remain. This paper enumerates the number of ways to break an $m \\times n$ bar, which we call chocolate numbers, and introduces four new sequences related to these numbers. Using various techniques, we p...

  15. Number theory

    CERN Document Server

    Andrews, George E

    1994-01-01

    Although mathematics majors are usually conversant with number theory by the time they have completed a course in abstract algebra, other undergraduates, especially those in education and the liberal arts, often need a more basic introduction to the topic.In this book the author solves the problem of maintaining the interest of students at both levels by offering a combinatorial approach to elementary number theory. In studying number theory from such a perspective, mathematics majors are spared repetition and provided with new insights, while other students benefit from the consequent simpl

  16. 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.

  17. Cas9, Cpf1 and C2c1/2/3-What's next?

    Science.gov (United States)

    Nakade, Shota; Yamamoto, Takashi; Sakuma, Tetsushi

    2017-05-04

    Since the rapid emergence of clustered regulatory interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system, developed as a genome engineering tool in 2012-2013, most researchers in the life science field have had a fixated interest in this fascinating technology. CRISPR-Cas9 is an RNA-guided DNA endonuclease system, which consists of Cas9 nuclease defining a few targeting base via protospacer adjacent motif complexed with easily customizable single guide RNA targeting around 20-bp genomic sequence. Although Streptococcus pyogenes Cas9 (SpCas9), one of the Cas9 proteins that applications in genome engineering were first demonstrated, still has wide usage because of its high nuclease activity and broad targeting range, there are several limitations such as large molecular weight and potential off-target effect. In this commentary, we describe various improvements and alternatives of CRISPR-Cas systems, including engineered Cas9 variants, Cas9 homologs, and novel Cas proteins other than Cas9. These variations enable flexible genome engineering with high efficiency and specificity, orthogonal genetic control at multiple gene loci, gene knockdown, or fluorescence imaging of transcripts mediated by RNA targeting, and beyond.

  18. The Revolution Continues: Newly Discovered Systems Expand the CRISPR-Cas Toolkit.

    Science.gov (United States)

    Murugan, Karthik; Babu, Kesavan; Sundaresan, Ramya; Rajan, Rakhi; Sashital, Dipali G

    2017-10-05

    CRISPR-Cas systems defend prokaryotes against bacteriophages and mobile genetic elements and serve as the basis for revolutionary tools for genetic engineering. Class 2 CRISPR-Cas systems use single Cas endonucleases paired with guide RNAs to cleave complementary nucleic acid targets, enabling programmable sequence-specific targeting with minimal machinery. Recent discoveries of previously unidentified CRISPR-Cas systems have uncovered a deep reservoir of potential biotechnological tools beyond the well-characterized Type II Cas9 systems. Here we review the current mechanistic understanding of newly discovered single-protein Cas endonucleases. Comparison of these Cas effectors reveals substantial mechanistic diversity, underscoring the phylogenetic divergence of related CRISPR-Cas systems. This diversity has enabled further expansion of CRISPR-Cas biotechnological toolkits, with wide-ranging applications from genome editing to diagnostic tools based on various Cas endonuclease activities. These advances highlight the exciting prospects for future tools based on the continually expanding set of CRISPR-Cas systems. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Structural Basis for the Altered PAM Specificities of Engineered CRISPR-Cas9.

    Science.gov (United States)

    Hirano, Seiichi; Nishimasu, Hiroshi; Ishitani, Ryuichiro; Nureki, Osamu

    2016-03-17

    The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets bearing a PAM (protospacer adjacent motif) and complementarity to the guide RNA. A recent study showed that, whereas wild-type Streptococcus pyogenes Cas9 (SpCas9) recognizes the 5'-NGG-3' PAM, the engineered VQR, EQR, and VRER SpCas9 variants recognize the 5'-NGA-3', 5'-NGAG-3', and 5'-NGCG-3' PAMs, respectively, thus expanding the targetable sequences in Cas9-mediated genome editing applications. Here, we present the high-resolution crystal structures of the three SpCas9 variants in complexes with a single-guide RNA and its altered PAM-containing, partially double-stranded DNA targets. A structural comparison of the three SpCas9 variants with wild-type SpCas9 revealed that the multiple mutations synergistically induce an unexpected displacement in the phosphodiester backbone of the PAM duplex, thereby allowing the SpCas9 variants to directly recognize the altered PAM nucleotides. Our findings explain the altered PAM specificities of the SpCas9 variants and establish a framework for further rational engineering of CRISPR-Cas9. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity.

    Science.gov (United States)

    Hu, Johnny H; Miller, Shannon M; Geurts, Maarten H; Tang, Weixin; Chen, Liwei; Sun, Ning; Zeina, Christina M; Gao, Xue; Rees, Holly A; Lin, Zhi; Liu, David R

    2018-04-05

    A key limitation of the use of the CRISPR-Cas9 system for genome editing and other applications is the requirement that a protospacer adjacent motif (PAM) be present at the target site. For the most commonly used Cas9 from Streptococcus pyogenes (SpCas9), the required PAM sequence is NGG. No natural or engineered Cas9 variants that have been shown to function efficiently in mammalian cells offer a PAM less restrictive than NGG. Here we use phage-assisted continuous evolution to evolve an expanded PAM SpCas9 variant (xCas9) that can recognize a broad range of PAM sequences including NG, GAA and GAT. The PAM compatibility of xCas9 is the broadest reported, to our knowledge, among Cas9 proteins that are active in mammalian cells, and supports applications in human cells including targeted transcriptional activation, nuclease-mediated gene disruption, and cytidine and adenine base editing. Notably, despite its broadened PAM compatibility, xCas9 has much greater DNA specificity than SpCas9, with substantially lower genome-wide off-target activity at all NGG target sites tested, as well as minimal off-target activity when targeting genomic sites with non-NGG PAMs. These findings expand the DNA targeting scope of CRISPR systems and establish that there is no necessary trade-off between Cas9 editing efficiency, PAM compatibility and DNA specificity.

  1. Chemical and Biophysical Modulation of Cas9 for Tunable Genome Engineering.

    Science.gov (United States)

    Nuñez, James K; Harrington, Lucas B; Doudna, Jennifer A

    2016-03-18

    The application of the CRISPR-Cas9 system for genome engineering has revolutionized the ability to interrogate genomes of mammalian cells. Programming the Cas9 endonuclease to induce DNA breaks at specified sites is achieved by simply modifying the sequence of its cognate guide RNA. Although Cas9-mediated genome editing has been shown to be highly specific, cleavage events at off-target sites have also been reported. Minimizing, and eventually abolishing, unwanted off-target cleavage remains a major goal of the CRISPR-Cas9 technology before its implementation for therapeutic use. Recent efforts have turned to chemical biology and biophysical approaches to engineer inducible genome editing systems for controlling Cas9 activity at the transcriptional and protein levels. Here, we review recent advancements to modulate Cas9-mediated genome editing by engineering split-Cas9 constructs, inteins, small molecules, protein-based dimerizing domains, and light-inducible systems.

  2. Methods for decoding Cas9 protospacer adjacent motif (PAM) sequences: A brief overview.

    Science.gov (United States)

    Karvelis, Tautvydas; Gasiunas, Giedrius; Siksnys, Virginijus

    2017-05-15

    Recently the Cas9, an RNA guided DNA endonuclease, emerged as a powerful tool for targeted genome manipulations. Cas9 protein can be reprogrammed to cleave, bind or nick any DNA target by simply changing crRNA sequence, however a short nucleotide sequence, termed PAM, is required to initiate crRNA hybridization to the DNA target. PAM sequence is recognized by Cas9 protein and must be determined experimentally for each Cas9 variant. Exploration of Cas9 orthologs could offer a diversity of PAM sequences and novel biochemical properties that may be beneficial for genome editing applications. Here we briefly review and compare Cas9 PAM identification assays that can be adopted for other PAM-dependent CRISPR-Cas systems. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. CRISPR-Cas and Contact-Dependent Secretion Systems Present on Excisable Pathogenicity Islands with Conserved Recombination Modules.

    Science.gov (United States)

    Carpenter, Megan R; Kalburge, Sai S; Borowski, Joseph D; Peters, Molly C; Colwell, Rita R; Boyd, E Fidelma

    2017-05-15

    Pathogenicity islands (PAIs) are mobile integrated genetic elements that contain a diverse range of virulence factors. PAIs integrate into the host chromosome at a tRNA locus that contains their specific bacterial attachment site, attB , via integrase-mediated site-specific recombination generating attL and attR sites. We identified conserved recombination modules (integrases and att sites) previously described in choleragenic Vibrio cholerae PAIs but with novel cargo genes. Clustered regularly interspaced short palindromic repeat (CRISPR)-associated proteins (Cas proteins) and a type VI secretion system (T6SS) gene cluster were identified at the Vibrio pathogenicity island 1 (VPI-1) insertion site in 19 V. cholerae strains and contained the same recombination module. Two divergent type I-F CRISPR-Cas systems were identified, which differed in Cas protein homology and content. The CRISPR repeat sequence was identical among all V. cholerae strains, but the CRISPR spacer sequences and the number of spacers varied. In silico analysis suggests that the CRISPR-Cas systems were active against phages and plasmids. A type III secretion system (T3SS) was present in 12 V. cholerae strains on a 68-kb island inserted at the same tRNA-serine insertion site as VPI-2 and contained the same recombination module. Bioinformatics analysis showed that two divergent T3SSs exist among the strains examined. Both the CRISPR and T3SS islands excised site specifically from the bacterial chromosome as complete units, and the cognate integrases were essential for this excision. These data demonstrated that identical recombination modules that catalyze integration and excision from the chromosome can acquire diverse cargo genes, signifying a novel method of acquisition for both CRISPR-Cas systems and T3SSs. IMPORTANCE This work demonstrated the presence of CRISPR-Cas systems and T3SSs on PAIs. Our work showed that similar recombination modules can associate with different cargo genes and

  4. Nice numbers

    CERN Document Server

    Barnes, John

    2016-01-01

    In this intriguing book, John Barnes takes us on a journey through aspects of numbers much as he took us on a geometrical journey in Gems of Geometry. Similarly originating from a series of lectures for adult students at Reading and Oxford University, this book touches a variety of amusing and fascinating topics regarding numbers and their uses both ancient and modern. The author intrigues and challenges his audience with both fundamental number topics such as prime numbers and cryptography, and themes of daily needs and pleasures such as counting one's assets, keeping track of time, and enjoying music. Puzzles and exercises at the end of each lecture offer additional inspiration, and numerous illustrations accompany the reader. Furthermore, a number of appendices provides in-depth insights into diverse topics such as Pascal’s triangle, the Rubik cube, Mersenne’s curious keyboards, and many others. A theme running through is the thought of what is our favourite number. Written in an engaging and witty sty...

  5. FACS-Assisted CRISPR-Cas9 Genome Editing Facilitates Parkinson's Disease Modeling

    Directory of Open Access Journals (Sweden)

    Jonathan Arias-Fuenzalida

    2017-11-01

    Full Text Available Genome editing and human induced pluripotent stem cells hold great promise for the development of isogenic disease models and the correction of disease-associated mutations for isogenic tissue therapy. CRISPR-Cas9 has emerged as a versatile and simple tool for engineering human cells for such purposes. However, the current protocols to derive genome-edited lines require the screening of a great number of clones to obtain one free of random integration or on-locus non-homologous end joining (NHEJ-containing alleles. Here, we describe an efficient method to derive biallelic genome-edited populations by the use of fluorescent markers. We call this technique FACS-assisted CRISPR-Cas9 editing (FACE. FACE allows the derivation of correctly edited polyclones carrying a positive selection fluorescent module and the exclusion of non-edited, random integrations and on-target allele NHEJ-containing cells. We derived a set of isogenic lines containing Parkinson's-disease-associated mutations in α-synuclein and present their comparative phenotypes.

  6. An astrosphere around the blue supergiant κ Cas: possible explanation of its filamentary structure

    Science.gov (United States)

    Katushkina, O. A.; Alexashov, D. B.; Gvaramadze, V. V.; Izmodenov, V. V.

    2018-01-01

    High-resolution mid-infrared observations carried out by the Spitzer Space Telescope allowed one to resolve the fine structure of many astrospheres. In particular, they showed that the astrosphere around the B0.7 Ia star κ Cas (HD 2905) has a clear-cut arc structure with numerous cirrus-like filaments beyond it. Previously, we suggested a physical mechanism for the formation of such filamentary structures. Namely, we showed theoretically that they might represent the non-monotonic spatial distribution of the interstellar dust in astrospheres (viewed as filaments) caused by interaction of the dust grains with the interstellar magnetic field disturbed in the astrosphere due to colliding of the stellar and interstellar winds. In this paper, we invoke this mechanism to explain the structure of the astrosphere around κ Cas. We performed 3D magnetohydrodynamic modelling of the astrosphere for realistic parameters of the stellar wind and space velocity. The dust dynamics and the density distribution in the astrosphere were calculated in the framework of a kinetic model. It is found that the model results with the classical MRN (Mathis, Rumpl & Nordsieck 1977) size distribution of dust in the interstellar medium do not match the observations, and that the observed filamentary structure of the astrosphere can be reproduced only if the dust is composed mainly of big (μm-sized) grains. Comparison of the model results with observations allowed us to estimate parameters (number density and magnetic field strength) of the surrounding interstellar medium.

  7. Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization. | Office of Cancer Genomics

    Science.gov (United States)

    The CRISPR/Cas9 system is a powerful tool for studying gene function. Here, we describe a method that allows temporal control of CRISPR/Cas9 activity based on conditional Cas9 destabilization. We demonstrate that fusing an FKBP12-derived destabilizing domain to Cas9 (DD-Cas9) enables conditional Cas9 expression and temporal control of gene editing in the presence of an FKBP12 synthetic ligand. This system can be easily adapted to co-express, from the same promoter, DD-Cas9 with any other gene of interest without co-modulation of the latter.

  8. Insights into the CRISPR/Cas system of Gardnerella vaginalis

    Directory of Open Access Journals (Sweden)

    Pleckaityte Milda

    2012-12-01

    Full Text Available Abstract Background Gardnerella vaginalis is identified as the predominant colonist of the vaginal tracts of women diagnosed with bacterial vaginosis (BV. G. vaginalis can be isolated from healthy women, and an asymptomatic BV state is also recognised. The association of G. vaginalis with different clinical phenotypes could be explained by different cytotoxicity of the strains, presumably based on disparate gene content. The contribution of horizontal gene transfer to shaping the genomes of G. vaginalis is acknowledged. The CRISPR loci of the recently discovered CRISPR/Cas microbial defence system provide a historical view of the exposure of prokaryotes to a variety of foreign genetic elements. Results The CRISPR/Cas loci were analysed using available sequence data from three G. vaginalis complete genomes and 18 G. vaginalis draft genomes in the NCBI database, as well as PCR amplicons of the genomic DNA of 17 clinical isolates. The cas genes in the CRISPR/Cas loci of G. vaginalis belong to the E. coli subtype. Approximately 20% of the spacers had matches in the GenBank database. Sequence analysis of the CRISPR arrays revealed that nearly half of the spacers matched G. vaginalis chromosomal sequences. The spacers that matched G. vaginalis chromosomal sequences were determined to not be self-targeting and were presumably neither constituents of mobile-element-associated genes nor derived from plasmids/viruses. The protospacers targeted by these spacers displayed conserved protospacer-adjacent motifs. Conclusions The CRISPR/Cas system has been identified in about one half of the analysed G. vaginalis strains. Our analysis of CRISPR sequences did not reveal a potential link between their presence and the virulence of the G. vaginalis strains. Based on the origins of the spacers found in the G. vaginalis CRISPR arrays, we hypothesise that the transfer of genetic material among G. vaginalis strains could be regulated by the CRISPR/Cas mechanism. The

  9. Number names and number understanding

    DEFF Research Database (Denmark)

    Ejersbo, Lisser Rye; Misfeldt, Morten

    2014-01-01

    This paper concerns the results from the first year of a three-year research project involving the relationship between Danish number names and their corresponding digits in the canonical base 10 system. The project aims to develop a system to help the students’ understanding of the base 10 syste...... the Danish number names are more complicated than in other languages. Keywords: A research project in grade 0 and 1th in a Danish school, Base-10 system, two-digit number names, semiotic, cognitive perspectives....

  10. Academic College of Emergency Experts in India′s INDO-US Joint Working Group (JWG White Paper on the Integrated Emergency Communication Response Service in India: Much more than just a number!

    Directory of Open Access Journals (Sweden)

    Anuja Joshi

    2013-01-01

    Full Text Available The proposal for an integrated national emergency number for India is garnering a lot of enthusiasm and stimulating debate. This ambitious project has a two-part paradigm shift to set in; the first being the integration into a single number and the infrastructure required for setting up and operating this number such that a call can be received and identified. The second is the submerged part of the iceberg: That of the ability to respond to a call and deliver the appropriate emergency service. The first part is more technical and has potential precedents like the 911 phone hotline, for example, to emulate. The main premise of this paper is that the second part is a rather subjective exercise largely determined by the realities of existing public infrastructure in a specific geographical area with respect to emergency services management, especially medical care. Consequently, we highlight the key areas of both precall preparedness and postcall execution that need to be reviewed prior to going live with an integrated number on a national scale.

  11. Funny Numbers

    Directory of Open Access Journals (Sweden)

    Theodore M. Porter

    2012-12-01

    Full Text Available The struggle over cure rate measures in nineteenth-century asylums provides an exemplary instance of how, when used for official assessments of institutions, these numbers become sites of contestation. The evasion of goals and corruption of measures tends to make these numbers “funny” in the sense of becoming dis-honest, while the mismatch between boring, technical appearances and cunning backstage manipulations supplies dark humor. The dangers are evident in recent efforts to decentralize the functions of governments and corporations using incen-tives based on quantified targets.

  12. Transcendental numbers

    CERN Document Server

    Murty, M Ram

    2014-01-01

    This book provides an introduction to the topic of transcendental numbers for upper-level undergraduate and graduate students. The text is constructed to support a full course on the subject, including descriptions of both relevant theorems and their applications. While the first part of the book focuses on introducing key concepts, the second part presents more complex material, including applications of Baker’s theorem, Schanuel’s conjecture, and Schneider’s theorem. These later chapters may be of interest to researchers interested in examining the relationship between transcendence and L-functions. Readers of this text should possess basic knowledge of complex analysis and elementary algebraic number theory.

  13. 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...

  14. Homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae.

    Science.gov (United States)

    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.

  15. Ouabain affects cell migration via Na,K-ATPase-p130cas and via nucleus-centrosome association.

    Directory of Open Access Journals (Sweden)

    Young Ou

    Full Text Available Na,K-ATPase is a membrane protein that catalyzes ATP to maintain transmembrane sodium and potassium gradients. In addition, Na,K-ATPase also acts as a signal-transducing receptor for cardiotonic steroids such as ouabain and activates a number of signalling pathways. Several studies report that ouabain affects cell migration. Here we used ouabain at concentrations far below those required to block Na,K-ATPase pump activity and show that it significantly reduced RPE cell migration through two mechanisms. It causes dephosphorylation of a 130 kD protein, which we identify as p130cas. Src is involved, because Src inhibitors, but not inhibitors of other kinases tested, caused a similar reduction in p130cas phosphorylation and ouabain increased the association of Na,K-ATPase and Src. Knockdown of p130cas by siRNA reduced cell migration. Unexpectedly, ouabain induced separation of nucleus and centrosome, also leading to a block in cell migration. Inhibitor and siRNA experiments show that this effect is mediated by ERK1,2. This is the first report showing that ouabain can regulate cell migration by affecting nucleus-centrosome association.

  16. Transfinite Numbers

    Indian Academy of Sciences (India)

    this is a characteristic difference between finite and infinite sets and created an immensely useful branch of mathematics based on this idea which had a great impact on the whole of mathe- matics. For example, the question of what is a number (finite or infinite) is almost a philosophical one. However Cantor's work turned it ...

  17. The development of Korea's new long-term care service infrastructure and its results: focusing on the market-friendly policy used for expansion of the numbers of service providers and personal care workers.

    Science.gov (United States)

    Chon, Yongho

    2013-01-01

    One of the main reasons for reforming long-term care systems is a deficient existing service infrastructure for the elderly. This article provides an overview of why and how the Korean government expanded long-term care infrastructure through the introduction of a new compulsory insurance system, with a particular focus on the market-friendly policies used to expand the infrastructure. Then, the positive results of the expansion of the long-term care infrastructure and the challenges that have emerged are examined. Finally, it is argued that the Korean government should actively implement a range of practical policies and interventions within the new system.

  18. 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.

  19. CRISPR/Cas-Mediated Knockin in Human Pluripotent Stem Cells.

    Science.gov (United States)

    Verma, Nipun; Zhu, Zengrong; Huangfu, Danwei

    2017-01-01

    Fluorescent reporter and epitope-tagged human pluripotent stem cells (hPSCs) greatly facilitate studies on the pluripotency and differentiation characteristics of these cells. Unfortunately traditional procedures to generate such lines are hampered by a low targeting efficiency that necessitates a lengthy process of selection followed by the removal of the selection cassette. Here we describe a procedure to generate fluorescent reporter and epitope tagged hPSCs in an efficient one-step process using the CRISPR/Cas technology. Although the method described uses our recently developed iCRISPR platform, the protocols can be adapted for general use with CRISPR/Cas or other engineered nucleases. The transfection procedures described could also be used for additional applications, such as overexpression or lineage tracing studies.

  20. CRISPR-Cas adaptive immune systems of the sulfolobales

    DEFF Research Database (Denmark)

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

    2015-01-01

    The Sulfolobales have provided good model organisms for studying CRISPR-Cas systems of the crenarchaeal kingdom of the archaea. These organisms are infected by a wide range of exceptional archaea-specific viruses and conjugative plasmids, and their CRISPR-Cas systems generally exhibit extensive...... structural and functional diversity. They carry large and multiple CRISPR loci and often multiple copies of diverse Type I and Type III interference modules as well as more homogeneous adaptation modules. These acidothermophilic organisms have recently provided seminal insights into both the adaptation...... process, the diverse modes of interference, and their modes of regulation. The functions of the adaptation and interference modules tend to be loosely coupled and the stringency of the crRNA-DNA sequence matching during DNA interference is relatively low, in contrast to some more streamlined CRISPR...

  1. CRISPR-Cas9 Toolkit for Actinomycete Genome Editing

    DEFF Research Database (Denmark)

    Tong, Yaojun; Robertsen, Helene Lunde; Blin, Kai

    2018-01-01

    engineering approaches for boosting known and discovering novel natural products. In order to facilitate the genome editing for actinomycetes, we developed a CRISPR-Cas9 toolkit with high efficiency for actinomyces genome editing. This basic toolkit includes a software for spacer (sgRNA) identification......, a system for in-frame gene/gene cluster knockout, a system for gene loss-of-function study, a system for generating a random size deletion library, and a system for gene knockdown. For the latter, a uracil-specific excision reagent (USER) cloning technology was adapted to simplify the CRISPR vector...... construction process. The application of this toolkit was successfully demonstrated by perturbation of genomes of Streptomyces coelicolor A3(2) and Streptomyces collinus Tü 365. The CRISPR-Cas9 toolkit and related protocol described here can be widely used for metabolic engineering of actinomycetes....

  2. A Scalable Framework and Prototype for CAS e-Science

    Directory of Open Access Journals (Sweden)

    Yuanchun Zhou

    2007-07-01

    Full Text Available Based on the Small-World model of CAS e-Science and the power low of Internet, this paper presents a scalable CAS e-Science Grid framework based on virtual region called Virtual Region Grid Framework (VRGF. VRGF takes virtual region and layer as logic manage-unit. In VRGF, the mode of intra-virtual region is pure P2P, and the model of inter-virtual region is centralized. Therefore, VRGF is decentralized framework with some P2P properties. Further more, VRGF is able to achieve satisfactory performance on resource organizing and locating at a small cost, and is well adapted to the complicated and dynamic features of scientific collaborations. We have implemented a demonstration VRGF based Grid prototype—SDG.

  3. Applying Registry Services to Spaceflight Technologies to Aid in the Assignment of Assigned Numbers to Disparate Systems and their Technologies to Further Enable Interoperability

    Science.gov (United States)

    Bradford, Robert N.; Nichols, Kelvin F.; Witherspoon, Keith R.

    2006-01-01

    To date very little effort has been made to provide interoperability between various space agency projects. To effectively get to the Moon and beyond systems must interoperate. To provide interoperability, standardization and registries of various technologies will be required. These registries will be created as they relate to space flight. With the new NASA Moon/Mars initiative, a requirement to standardize and control the naming conventions of very disparate systems and technologies is emerging. The need to provide numbering to the many processes, schemas, vehicles, robots, space suits and technologies (e.g. versions), to name a few, in the highly complex Constellation initiative is imperative. The number of corporations, developer personnel, system interfaces, people interfaces will require standardization and registries on a scale not currently envisioned. It would only take one exception (stove piped system development) to weaken, if not, destroy interoperability. To start, a standardized registry process must be defined that allows many differing engineers, organizations and operators the ability to easily access disparate registry information across numerous technological and scientific disciplines. Once registries are standardized the need to provide registry support in terms of setup and operations, resolution of conflicts between registries and other issues will need to be addressed. Registries should not be confused with repositories. No end user data is "stored" in a registry nor is it a configuration control system. Once a registry standard is created and approved, the technologies that should be registered must be identified and prioritized. In this paper, we will identify and define a registry process that is compatible with the Constellation initiative and other non related space activities and organizations. We will then identify and define the various technologies that should use a registry to provide interoperability. The first set of

  4. Excision of Nucleopolyhedrovirus Form Transgenic Silkworm Using the CRISPR/Cas9 System

    Directory of Open Access Journals (Sweden)

    Zhanqi Dong

    2018-02-01

    Full Text Available The CRISPR/Cas9-mediated genome engineering has been shown to efficiently suppress infection by disrupting genes of the pathogen. We recently constructed transgenic lines expressing CRISPR/Cas9 and the double sgRNA target Bombyx mori nucleopolyhedrovirus (BmNPV immediate early-1 (ie-1 gene in the silkworm, respectively, and obtained four transgenic hybrid lines by G1 generation hybridization: Cas9(-/sgRNA(-, Cas9(+/sgRNA(-, Cas9(-/sgRNA(+, and Cas9(+/sgRNA(+. We demonstrated that the Cas9(+/sgRNA(+ transgenic lines effectively edited the target site of the BmNPV genome, and large fragment deletion was observed after BmNPV infection. Further antiviral analysis of the Cas9(+/sgRNA(+ transgenic lines shows that the median lethal dose (LD50 is 1,000-fold higher than the normal lines after inoculation with occlusion bodies. The analysis of economic characters and off-target efficiency of Cas9(+/sgRNA(+ transgenic hybrid line showed no significant difference compared with the normal lines. Our findings indicate that CRISPR/Cas9-mediated genome engineering more effectively targets the BmNPV genomes and could be utilized as an insect antiviral treatment.

  5. Excision of Nucleopolyhedrovirus Form Transgenic Silkworm Using the CRISPR/Cas9 System.

    Science.gov (United States)

    Dong, Zhanqi; Dong, Feifan; Yu, Xinbo; Huang, Liang; Jiang, Yaming; Hu, Zhigang; Chen, Peng; Lu, Cheng; Pan, Minhui

    2018-01-01

    The CRISPR/Cas9-mediated genome engineering has been shown to efficiently suppress infection by disrupting genes of the pathogen. We recently constructed transgenic lines expressing CRISPR/Cas9 and the double sgRNA target Bombyx mori nucleopolyhedrovirus (BmNPV) immediate early-1 ( ie-1 ) gene in the silkworm, respectively, and obtained four transgenic hybrid lines by G1 generation hybridization: Cas9(-)/sgRNA(-), Cas9(+)/sgRNA(-), Cas9(-)/sgRNA(+), and Cas9(+)/sgRNA(+). We demonstrated that the Cas9(+)/sgRNA(+) transgenic lines effectively edited the target site of the BmNPV genome, and large fragment deletion was observed after BmNPV infection. Further antiviral analysis of the Cas9(+)/sgRNA(+) transgenic lines shows that the median lethal dose (LD50) is 1,000-fold higher than the normal lines after inoculation with occlusion bodies. The analysis of economic characters and off-target efficiency of Cas9(+)/sgRNA(+) transgenic hybrid line showed no significant difference compared with the normal lines. Our findings indicate that CRISPR/Cas9-mediated genome engineering more effectively targets the BmNPV genomes and could be utilized as an insect antiviral treatment.

  6. Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications.

    Science.gov (United States)

    Liu, Chang; Zhang, Li; Liu, Hao; Cheng, Kun

    2017-11-28

    The CRISPR-Cas9 genome-editing system is a part of the adaptive immune system in archaea and bacteria to defend against invasive nucleic acids from phages and plasmids. The single guide RNA (sgRNA) of the system recognizes its target sequence in the genome, and the Cas9 nuclease of the system acts as a pair of scissors to cleave the double strands of DNA. Since its discovery, CRISPR-Cas9 has become the most robust platform for genome engineering in eukaryotic cells. Recently, the CRISPR-Cas9 system has triggered enormous interest in therapeutic applications. CRISPR-Cas9 can be applied to correct disease-causing gene mutations or engineer T cells for cancer immunotherapy. The first clinical trial using the CRISPR-Cas9 technology was conducted in 2016. Despite the great promise of the CRISPR-Cas9 technology, several challenges remain to be tackled before its successful applications for human patients. The greatest challenge is the safe and efficient delivery of the CRISPR-Cas9 genome-editing system to target cells in human body. In this review, we will introduce the molecular mechanism and different strategies to edit genes using the CRISPR-Cas9 system. We will then highlight the current systems that have been developed to deliver CRISPR-Cas9 in vitro and in vivo for various therapeutic purposes. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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.

  8. 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

  9. 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

  10. Cas9-nickase-mediated genome editing corrects hereditary tyrosinemia in rats.

    Science.gov (United States)

    Shao, Yanjiao; Wang, Liren; Guo, Nana; Wang, Shengfei; Yang, Lei; Li, Yajing; Wang, Mingsong; Yin, Shuming; Han, Honghui; Zeng, Li; Zhang, Ludi; Hui, Lijian; Ding, Qiurong; Zhang, Jiqin; Geng, Hongquan; Liu, Mingyao; Li, Dali

    2018-05-04

    Hereditary tyrosinemia type I (HTI) is a metabolic genetic disorder caused by mutation of fumarylacetoacetate hydrolase (FAH). Because of the accumulation of toxic metabolites, HTI causes severe liver cirrhosis, liver failure, and even hepatocellular carcinoma. HTI is an ideal model for gene therapy, and several strategies have been shown to ameliorate HTI symptoms in animal models. Although CRISPR/Cas9-mediated genome editing is able to correct the Fah mutation in mouse models, WT Cas9 induces numerous undesired mutations that have raised safety concerns for clinical applications. To develop a new method for gene correction with high fidelity, we generated a Fah mutant rat model to investigate whether Cas9 nickase (Cas9n)-mediated genome editing can efficiently correct the Fah First, we confirmed that Cas9n rarely induces indels in both on-target and off-target sites in cell lines. Using WT Cas9 as a positive control, we delivered Cas9n and the repair donor template/single guide (sg)RNA through adenoviral vectors into HTI rats. Analyses of the initial genome editing efficiency indicated that only WT Cas9 but not Cas9n causes indels at the on-target site in the liver tissue. After receiving either Cas9n or WT Cas9-mediated gene correction therapy, HTI rats gained weight steadily and survived. Fah-expressing hepatocytes occupied over 95% of the liver tissue 9 months after the treatment. Moreover, CRISPR/Cas9-mediated gene therapy prevented the progression of liver cirrhosis, a phenotype that could not be recapitulated in the HTI mouse model. These results strongly suggest that Cas9n-mediated genome editing is a valuable and safe gene therapy strategy for this genetic disease. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Evolution of resistance against CRISPR/Cas9 gene drive

    OpenAIRE

    Clark, Andrew; Unckless, Robert; Messer, Philipp

    2016-01-01

    CRISPR/Cas9 gene drive (CGD) promises to be a highly adaptable approach for spreading genetically engineered alleles throughout a species, even if those alleles impair reproductive success. CGD has been shown to be effective in laboratory crosses of insects, yet it remains unclear to what extent potential resistance mechanisms will affect the dynamics of this process in large natural populations. Here we develop a comprehensive population genetic framework for modeling CGD dynamics, which inc...

  12. Communications en cas de catastrophe faisant appel aux TIC pour ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    Communications en cas de catastrophe faisant appel aux TIC pour les collectivités vulnérables des Caraïbes. De récents événements survenus dans les Caraïbes ont mis en relief les insuffisances des mesures régionales et nationales de préparation aux catastrophes. On manque particulièrement de systèmes d'alerte ...

  13. Application of the nanobiotechnology with the system CRISP-Cas

    OpenAIRE

    Liceth Xiomara Sáenz-Castiblanco; Maritza Angarita-Merchán; Diana Paola Lopez-Velandia

    2017-01-01

    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. Materia...

  14. Cas d'une loi exponentielle Bayesian predict

    African Journals Online (AJOL)

    DK

    Avec des données groupées : Cas d'une loi exponentielle. Bayesian predictions of order statistics with grouped data: The case of an exponential law. Assia Chadli* & Asma Meradji. Laboratoire LaPS, Département de Mathématiques, Faculté des Sciences. Université Badji Mokhtar Annaba, BP 12, 23000, Annaba, Algérie.

  15. On a strong law of large numbers for monotone measures

    Czech Academy of Sciences Publication Activity Database

    Agahi, H.; Mohammadpour, A.; Mesiar, Radko; Ouyang, Y.

    2013-01-01

    Roč. 83, č. 4 (2013), s. 1213-1218 ISSN 0167-7152 R&D Projects: GA ČR GAP402/11/0378 Institutional support: RVO:67985556 Keywords : capacity * Choquet integral * strong law of large numbers Subject RIV: BA - General Mathematics Impact factor: 0.531, year: 2013 http://library.utia.cas.cz/separaty/2013/E/mesiar-on a strong law of large numbers for monotone measures.pdf

  16. Determination of the number of ψ(3686) events at BESIII

    Science.gov (United States)

    Ablikim, M.; Achasov, M. N.; Ai, X. C.; Ambrose, D. J.; Amoroso, A.; An, F. F.; An, Q.; Bai, J. Z.; Baldini Ferroli, R.; Ban, Y.; Bennett, J. V.; Bertani, M.; Bian, J. M.; Boger, E.; Bondarenko, O.; Boyko, I.; Briere, R. A.; Cai, H.; Cai, X.; Cakir, O.; Calcaterra, A.; Cao, G. F.; Cetin, S. A.; Chang, J. F.; Chelkov, G.; Chen, G.; Chen, H. S.; Chen, J. C.; Chen, M. L.; Chen, S. J.; Chen, X.; Chen, X. R.; Chen, Y. B.; Chu, X. K.; Chu, Y. P.; Cronin-Hennessy, D.; Dai, H. L.; Dai, J. P.; Dedovich, D.; Deng, Z. Y.; Denig, A.; Denysenko, I.; Destefanis, M.; Ding, Y.; Dong, C.; Dong, J.; Dong, L. Y.; Dong, M. Y.; Du, S. X.; Fan, J. Z.; Fang, J.; Fang, S. S.; Fang, Y.; Fava, L.; Feldbauer, F.; Feng, C. Q.; Fu, C. D.; Gao, Q.; Gao, Y.; Goetzen, K.; Gong, W. X.; Gradl, W.; Greco, M.; Gu, M. H.; Gu, Y. T.; Guan, Y. H.; Guo, A. Q.; Guo, Y. P.; Han, Y. L.; Harris, F. A.; He, K. L.; He, M.; Held, T.; Heng, Y. K.; Hou, Z. L.; Hu, H. M.; Hu, T.; Huang, G. S.; Huang, J. S.; Huang, L.; Huang, X. T.; Hussain, T.; Ji, Q.; Ji, Q. P.; Ji, X. B.; Ji, X. L.; Jiang, L. L.; Jiang, X. S.; Jiao, J. B.; Jiao, Z.; Jin, D. P.; Jin, S.; Johansson, T.; Kalantar-Nayestanaki, N.; Kang, X. L.; Kang, X. S.; Kavatsyuk, M.; Kloss, B.; Kopf, B.; Kornicer, M.; Kupsc, A.; Kühn, W.; Lai, W.; Lange, J. S.; Lara, M.; Larin, P.; Li, C. H.; Li, Cheng; Li, D. M.; Li, F.; Li, G.; Li, H. B.; Li, J. C.; Li, Kang; Li, Ke; Li, Lei; Li, P. R.; Li, Q. J.; Li, W. D.; Li, W. G.; Li, X. L.; Li, X. N.; Li, X. Q.; Li, X. R.; Li, Z. B.; Liang, H.; Liang, Y. F.; Liang, Y. T.; Liao, G. R.; Lin, D. X.; Liu, B. J.; Liu, C. X.; Liu, F. H.; Liu, Fang.; Liu, Feng.; Liu, H. B.; Liu, H. M.; Liu, Huihui.; Liu, J.; Liu, J. P.; Liu, K.; Liu, K. Y.; Liu, Q.; Liu, S. B.; Liu, X.; Liu, Y. B.; Liu, Z. A.; Liu, Zhiqiang.; Liu, Zhiqing.; Loehner, H.; Lou, X. C.; Lu, H. J.; Lu, H. L.; Lu, J. G.; Lu, Y.; Lu, Y. P.; Luo, C. L.; Luo, M. X.; Luo, T.; Luo, X. L.; Lv, M.; Lyu, X. R.; Ma, F. C.; Ma, H. L.; Ma, Q. M.; Ma, S.; Ma, T.; Ma, X. Y.; Maas, F. E.; Maggiora, M.; Mao, Y. J.; Mao, Z. P.; Messchendorp, J. G.; Min, J.; Min, T. J.; Mitchell, R. E.; Mo, X. H.; Mo, Y. J.; Morales Morales, C.; Moriya, K.; Muchnoi, N. Yu.; Muramatsu, H.; Nefedov, Y.; Nikolaev, I. B.; Ning, Z.; Nisar, S.; Niu, S. L.; Niu, X. Y.; Olsen, S. L.; Ouyang, Q.; Pacetti, S.; Pelizaeus, M.; Peng, H. P.; Peters, K.; Ping, J. L.; Ping, R. G.; Poling, R.; Qi, M.; Qian, S.; Qiao, C. F.; Qin, X. S.; Qin, Z. H.; Qiu, J. F.; Rashid, K. H.; Redmer, C. F.; Ripka, M.; Rong, G.; Sarantsev, A.; Schoenning, K.; Shan, W.; Shao, M.; Shen, C. P.; Shen, X. Y.; Sheng, H. Y.; Shepherd, M. R.; Song, W. M.; Song, X. Y.; Sosio, S.; Spataro, S.; Sun, G. X.; Sun, J. F.; Sun, S. S.; Sun, Y. J.; Sun, Y. Z.; Sun, Z. J.; Tang, C. J.; Tang, X.; Tapan, I.; Thorndike, E. H.; Toth, D.; Uman, I.; Varner, G. S.; Wang, B.; Wang, D.; Wang, D. Y.; Wang, K.; Wang, L. L.; Wang, L. S.; Wang, M.; Wang, P.; Wang, P. L.; Wang, Q. J.; Wang, W.; Wang, X. F.; Wang(Yadi, Y. D.; Wang, Y. F.; Wang, Y. Q.; Wang, Z.; Wang, Z. G.; Wang, Z. Y.; Wei, D. H.; Weidenkaff, P.; Wen, S. P.; Wiedner, U.; Wolke, M.; Wu, L. H.; Wu, Z.; Xia, L. G.; Xia, Y.; Xiao, D.; Xiao, Z. J.; Xie, Y. G.; Xiu, Q. L.; Xu, G. F.; Xu, L.; Xu, Q. J.; Xu, Q. N.; Xu, X. P.; Yan, W. B.; Yan, Y. H.; Yang, H. X.; Yang, Y.; Yang, Y. X.; Ye, H.; Ye, M.; Ye, M. H.; Yu, B. X.; Yu, C. X.; Yu, J. S.; Yuan, C. Z.; Yuan, Y.; Zafar, A. A.; Zeng, Y.; Zhang, B. X.; Zhang, B. Y.; Zhang, C. C.; Zhang, D. H.; Zhang, H. H.; Zhang, H. Y.; Zhang, J. J.; Zhang, J. Q.; Zhang, J. W.; Zhang, J. Y.; Zhang, J. Z.; Zhang, L.; Zhang, R.; Zhang, S. H.; Zhang, X. J.; Zhang, X. Y.; Zhang, Y. H.; Zhang, Yao.; Zhang, Z. H.; Zhang, Z. P.; Zhang, Z. Y.; Zhao, G.; Zhao, J. W.; Zhao, J. Z.; Zhao, Lei; Zhao, Ling.; Zhao, M. G.; Zhao, Q.; Zhao, Q. W.; Zhao, S. J.; Zhao, T. C.; Zhao, Y. B.; Zhao, Z. G.; Zhemchugov, A.; Zheng, B.; Zheng, J. P.; Zheng, Y. H.; Zhong, B.; Zhou, L.; Zhou, X.; Zhou, X. K.; Zhou, X. R.; Zhou, X. Y.; Zhu, K.; Zhu, K. J.; Zhu, X. L.; Zhu, Y. C.; Zhu, Y. S.; Zhu, Z. A.; Zhuang, J.; Zou, B. S.; Zou, J. H.; BESIII Collaboration

    2018-02-01

    The numbers of ψ(3686) events accumulated by the BESIII detector for the data taken during 2009 and 2012 are determined to be (107.0+/- 0.8)× {10}6 and (341.1+/- 2.1)× {10}6, respectively, by counting inclusive hadronic events, where the uncertainties are systematic and the statistical uncertainties are negligible. The number of events for the sample taken in 2009 is consistent with that of the previous measurement. The total number of ψ(3686) events for the two data taking periods is (448.1+/- 2.9)× {10}6. Supported by the Ministry of Science and Technology of China (2009CB825200), National Natural Science Foundation of China (NSFC) (11235011, 11322544, 11335008, 11425524, 11475207), the Chinese Academy of Sciences (CAS) Large-Scale Scientific Facility Program, the Collaborative Innovation Center for Particles and Interactions (CICPI), Joint Large-Scale Scientific Facility Funds of the NSFC and CAS (11179014), Joint Large-Scale Scientific Facility Funds of the NSFC and CAS (11179007, U1232201, U1532257, U1532258), Joint Funds of the National Natural Science Foundation of China (11079008), CAS (KJCX2-YW-N29, KJCX2-YW-N45), 100 Talents Program of CAS, National 1000 Talents Program of China, German Research Foundation DFG (Collaborative Research Center CRC 1044), Istituto Nazionale di Fisica Nucleare, Italy, Koninklijke Nederlandse Akademie van Wetenschappen (KNAW) (530-4CDP03), Ministry of Development of Turkey (DPT2006K-120470), National Natural Science Foundation of China (11205082), The Swedish Research Council, U. S. Department of Energy (DE-FG02-05ER41374, DE-SC-0010118, DE-SC-0010504), U.S. National Science Foundation, University of Groningen (RuG) and the Helmholtzzentrum fuer Schwerionenforschung GmbH (GSI), Darmstadt, WCU Program of National Research Foundation of Korea (R32-2008-000-10155-0).

  17. 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.

  18. 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.

  19. CRISPR/Cas-mediated targeted mutagenesis in Daphnia magna.

    Directory of Open Access Journals (Sweden)

    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.

  20. Le syndrome néphrotique idiopathique (SNI) de l’enfant à Dakar: à propos de 40 cas

    Science.gov (United States)

    Keita, Younoussa; Lemrabott, Ahmed Tall; Sylla, Assane; Niang, Babacar; Ka, El Hadji Fary; Dial, Chérif Mohamed; Ndongo, Aliou Abdoulaye; Sow, Amadou; Moreira, Claude; Niang, Abdou; Ndiaye, Ousmane; Diouf, Boucar; Sall, Mouhamadou Guélaye

    2017-01-01

    Introduction L’objectif de ce travail était d’analyser les caractéristiques diagnostiques, thérapeutiques et évolutives de l’enfant atteint de néphrose dans un service de pédiatrie de Dakar. Méthodes L’étude était réalisée au service de pédiatrie de l’hôpital Aristide Le Dantec. Il s’agissait d’une étude rétrospective sur une période de 03 ans allant du 1er janvier 2012 au 31 décembre 2014. Ont été inclus tous les patients âgés de 02 ans à 12 ans présentant un tableau de Syndrome néphrotique idiopathique. Résultats Quarante cas de néphrose étaient colligés soit une prévalence de 23% parmi les néphropathies prises en charge dans le service. L’âge moyen était de 7,11± 3,14 ans. Le syndrome néphrotique était pur chez 72,5% (n=29) des patients. Les œdèmes des membres inférieurs étaient présents chez 100% des patients, l’oligurie dans 55% (n=22) et l’HTA dans 5% (n=2) des cas. La protéinurie moyenne était de 145,05 ± 85,54 mg/kg/24heures. La protidémie moyenne était de 46,42 ±7,88 g/L et l’albuminémie moyenne de 17,90 ± 7,15 g/L. Trente-neuf patients avaient reçu une corticothérapie à base de prednisone. La corticosensibilité était retenue chez 77% (n=30) des patients et la corticorésistance chez 13% (n=5) des cas. Le facteur de mauvaise réponse à la corticothérapie était un niveau de protéinurie initiale supérieure à 150 mg/kg/jour (p = 0,024). La biopsie rénale était réalisée chez 18% (n=7) des patients et retrouvait dans 57,2% (n=4) des cas une hyalinose segmentaire et focale. Le cyclophosphamide et l’azathioprine étaient associés aux corticoïdes dans 10% (n=4) des cas chacun. Le taux de rémission globale était de 89,8%. L’évolution vers l’insuffisance rénale chronique était notée chez trois (03) des patients. Conclusion La néphrose représentait près du quart des néphropathies prises en charge dans notre service. Le taux de rémission globale était élevé. Le

  1. CRISPR-Cas Systems in Bacteroides fragilis, an Important Pathobiont in the Human Gut Microbiome

    Science.gov (United States)

    Tajkarimi, Mehrdad; Wexler, Hannah M.

    2017-01-01

    Background: While CRISPR-Cas systems have been identified in bacteria from a wide variety of ecological niches, there are no studies to describe CRISPR-Cas elements in Bacteroides species, the most prevalent anaerobic bacteria in the lower intestinal tract. Microbes of the genus Bacteroides make up ~25% of the total gut microbiome. Bacteroides fragilis comprises only 2% of the total Bacteroides in the gut, yet causes of >70% of Bacteroides infections. The factors causing it to transition from benign resident of the gut microbiome to virulent pathogen are not well understood, but a combination of horizontal gene transfer (HGT) of virulence genes and differential transcription of endogenous genes are clearly involved. The CRISPR-Cas system is a multi-functional system described in prokaryotes that may be involved in control both of HGT and of gene regulation. Results: Clustered regularly interspaced short palindromic repeats (CRISPR) elements in all strains of B. fragilis (n = 109) with publically available genomes were identified. Three different CRISPR-Cas types, corresponding most closely to Type IB, Type IIIB, and Type IIC, were identified. Thirty-five strains had two CRISPR-Cas types, and three strains included all three CRISPR-Cas types in their respective genomes. The cas1 gene in the Type IIIB system encoded a reverse-transcriptase/Cas1 fusion protein rarely found in prokaryotes. We identified a short CRISPR (3 DR) with no associated cas genes present in most of the isolates; these CRISPRs were found immediately upstream of a hipA/hipB operon and we speculate that this element may be involved in regulation of this operon related to formation of persister cells during antimicrobial exposure. Also, blood isolates of B. fragilis did not have Type IIC CRISPR-Cas systems and had atypical Type IIIB CRISPR-Cas systems that were lacking adjacent cas genes. Conclusions: This is the first systematic report of CRISPR-Cas systems in a wide range of B. fragilis strains

  2. CRISPR-Cas Systems in Bacteroides fragilis, an Important Pathobiont in the Human Gut Microbiome

    Directory of Open Access Journals (Sweden)

    Mehrdad Tajkarimi

    2017-11-01

    Full Text Available Background: While CRISPR-Cas systems have been identified in bacteria from a wide variety of ecological niches, there are no studies to describe CRISPR-Cas elements in Bacteroides species, the most prevalent anaerobic bacteria in the lower intestinal tract. Microbes of the genus Bacteroides make up ~25% of the total gut microbiome. Bacteroides fragilis comprises only 2% of the total Bacteroides in the gut, yet causes of >70% of Bacteroides infections. The factors causing it to transition from benign resident of the gut microbiome to virulent pathogen are not well understood, but a combination of horizontal gene transfer (HGT of virulence genes and differential transcription of endogenous genes are clearly involved. The CRISPR-Cas system is a multi-functional system described in prokaryotes that may be involved in control both of HGT and of gene regulation.Results: Clustered regularly interspaced short palindromic repeats (CRISPR elements in all strains of B. fragilis (n = 109 with publically available genomes were identified. Three different CRISPR-Cas types, corresponding most closely to Type IB, Type IIIB, and Type IIC, were identified. Thirty-five strains had two CRISPR-Cas types, and three strains included all three CRISPR-Cas types in their respective genomes. The cas1 gene in the Type IIIB system encoded a reverse-transcriptase/Cas1 fusion protein rarely found in prokaryotes. We identified a short CRISPR (3 DR with no associated cas genes present in most of the isolates; these CRISPRs were found immediately upstream of a hipA/hipB operon and we speculate that this element may be involved in regulation of this operon related to formation of persister cells during antimicrobial exposure. Also, blood isolates of B. fragilis did not have Type IIC CRISPR-Cas systems and had atypical Type IIIB CRISPR-Cas systems that were lacking adjacent cas genes.Conclusions: This is the first systematic report of CRISPR-Cas systems in a wide range of B

  3. 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.

  4. CRISPR/Cas9-mediated targeted gene correction in amyotrophic lateral sclerosis patient iPSCs.

    Science.gov (United States)

    Wang, Lixia; Yi, Fei; Fu, Lina; Yang, Jiping; Wang, Si; Wang, Zhaoxia; Suzuki, Keiichiro; Sun, Liang; Xu, Xiuling; Yu, Yang; Qiao, Jie; Belmonte, Juan Carlos Izpisua; Yang, Ze; Yuan, Yun; Qu, Jing; Liu, Guang-Hui

    2017-05-01

    Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disease with cellular and molecular mechanisms yet to be fully described. Mutations in a number of genes including SOD1 and FUS are associated with familial ALS. Here we report the generation of induced pluripotent stem cells (iPSCs) from fibroblasts of familial ALS patients bearing SOD1 +/A272C and FUS +/G1566A mutations, respectively. We further generated gene corrected ALS iPSCs using CRISPR/Cas9 system. Genome-wide RNA sequencing (RNA-seq) analysis of motor neurons derived from SOD1 +/A272C and corrected iPSCs revealed 899 aberrant transcripts. Our work may shed light on discovery of early biomarkers and pathways dysregulated in ALS, as well as provide a basis for novel therapeutic strategies to treat ALS.

  5. CRISPR-Cas9; an efficient tool for precise plant genome editing.

    Science.gov (United States)

    Islam, Waqar

    2018-04-03

    Efficient plant genome editing is dependent upon induction of double stranded DNA breaks (DSBs) through site specified nucleases. These DSBs initiate the process of DNA repair which can either base upon homologous recombination (HR) or non-homologous end jointing (NHEJ). Recently, CRISPR-Cas9 mechanism got highlighted as revolutionizing genetic tool due to its simpler frame work along with the broad range of adaptability and applications. So, in this review, I have tried to sum up the application of this biotechnological tool in plant genome editing. Furthermore, I have tried to explain successful adaptation of CRISPR in various plant species where it is used for the successful generation of stable mutations in a steadily growing number of species through NHEJ. The review also sheds light upon other biotechnological approaches relying upon single DNA lesion induction such as genomic deletion or pair wise nickases for evasion of offsite effects. Copyright © 2018 Elsevier Ltd. All rights reserved.

  6. CRISPR/Cas9-mediated targeted gene correction in amyotrophic lateral sclerosis patient iPSCs

    Directory of Open Access Journals (Sweden)

    Lixia Wang

    2017-04-01

    Full Text Available Abstract Amyotrophic lateral sclerosis (ALS is a complex neurodegenerative disease with cellular and molecular mechanisms yet to be fully described. Mutations in a number of genes including SOD1 and FUS are associated with familial ALS. Here we report the generation of induced pluripotent stem cells (iPSCs from fibroblasts of familial ALS patients bearing SOD1 +/A272C and FUS +/G1566A mutations, respectively. We further generated gene corrected ALS iPSCs using CRISPR/Cas9 system. Genome-wide RNA sequencing (RNA-seq analysis of motor neurons derived from SOD1 +/A272C and corrected iPSCs revealed 899 aberrant transcripts. Our work may shed light on discovery of early biomarkers and pathways dysregulated in ALS, as well as provide a basis for novel therapeutic strategies to treat ALS.

  7. Digoeuvre van Cas Vos: die beliggaming van �n lewensvreugdespel

    Directory of Open Access Journals (Sweden)

    J Buscop

    2003-10-01

    Full Text Available Since� 1999 Cas Vos has produced three books of poetry; Vuurtong (1999, Gode van Papier (2001 and Enkeldiep (2003. These works are all written from the perspective of� a� particular� Weltanschauung or Zeitgeist which may be defined as La joie de vivre. Joy of life manifests itself on a number of levels, including the Greek, pneuma, psuche and soma. La joie de vivre is also a crucial dimension of the gameplay that is love. Vos writes and lives love as a gift of God and identifies three stages of love, the Greek, eros, phileo and agape love. Whilst the joy of life has many sides, Vos also indicates� the� opposite manifestations of joie de vivre in pain, sickness, loss and death. Vos is a new inspiration in Afrikaans literature and has created an idiolectic voice which, in his own words, can be summarized as the coitus between different language signs.

  8. RNA-guided Transcriptional Regulation in Plants via dCas9 Chimeric Proteins

    KAUST Repository

    Baazim, Hatoon

    2014-05-01

    Developing targeted genome regulation approaches holds much promise for accelerating trait discovery and development in agricultural biotechnology. Clustered Regularly Interspaced Palindromic Repeats (CRISPRs)/CRISPR associated (Cas) system provides bacteria and archaea with an adaptive molecular immunity mechanism against invading nucleic acids through phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing purposes across a variety of cell types and organisms. Recently, the catalytically inactive Cas9 (dCas9) protein combined with guide RNAs (gRNAs) were used as a DNA-targeting platform to modulate the expression patterns in bacterial, yeast and human cells. Here, we employed this DNA-targeting system for targeted transcriptional regulation in planta by developing chimeric dCas9-based activators and repressors. For example, we fused to the C-terminus of dCas9 with the activation domains of EDLL and TAL effectors, respectively, to generate transcriptional activators, and the SRDX repression domain to generate transcriptional repressor. Our data demonstrate that the dCas9:EDLL and dCas9:TAD activators, guided by gRNAs complementary to promoter elements, induce strong transcriptional activation on episomal targets in plant cells. Moreover, our data suggest that the dCas9:SRDX repressor and the dCas9:EDLL and dCas9:TAD activators are capable of markedly repressing or activating, respectively, the transcription of an endogenous genomic target. Our data indicate that the CRISPR/dCas9:TFs DNA targeting system can be used in plants as a functional genomic tool and for biotechnological applications.

  9. 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....

  10. Modelling of slag emulsification and slag reduction in CAS-OB process

    OpenAIRE

    Sulasalmi, P. (Petri)

    2016-01-01

    Abstract Composition Adjustment by Sealed argon bubbling – Oxygen Blowing (CAS-OB) process is a ladle treatment process that was developed for chemical heating and alloying of steel. The main stages of the process are heating, (possible) alloying and reduction of slag. The CAS-OB process aims for homogenization and control of the composition and temperature of steel. In this dissertation, a mathematical reaction model was developed for the slag reduction stage of the CAS-OB process. Sl...

  11. Les leishmanioses cutanées à Leishmania major et à Leishmania tropica au Maroc: aspects épidémio-cliniques comparatifs de 268 cas

    Science.gov (United States)

    Chiheb, Soumia; Slaoui, Widad; Mouttaqui, Tarik; Riyad, Meriem; Benchikhi, Hakima

    2014-01-01

    Introduction Depuis 1995, le Maroc a connu une réactivation des foyers de leishmanioses cutanées (LC) à L. major et une nouvelle répartition géographique des foyers à L. tropica. Le but de cette étude est de comparer les aspects épidémio-cliniques associés aux LC potentiellement dûes à L. major et à L. tropica. Méthodes Une étude rétrospective a colligé 268 cas de LC au service de dermatologie du CHU Ibn Rochd de Casablanca entre Janvier 1995 et Septembre 2010. Les données étaient analysées par Epi info version 3.5.1. Le test X2 était appliqué (Différence significative = p< 0,05). Résultats Deux cent soixante-huit cas de LC ont été colligés, dont 160 femmes et 108 hommes. Ils ont été répartis en 123 patients originaires des foyers à L.major et 145 patients originaires des foyers à L. tropica. L'aspect ulcéronodulaire, ulcérovégétant ou végétant était retrouvé dans 58 cas (47,2%) des cas de LC à L. major versus 24 cas (16,7%) dans la L.C à L. tropica. L'aspect papulonodulaire était retrouvé dans 84 cas (58%) de LC à L. tropica contre 41 cas (33,3%) de LC à L. major. Conclusion Dans la LC à L. major, l'atteinte des membres et les aspects cliniques végétant ou ulcéro-végétant restent toujours prédominants. Dans la L.C à L. tropica, l'atteinte papulonodulaire unique du visage reste prédominante mais des formes ulcéronodulaires, végétantes ou ulcérovégétantes existent également dans les foyers récents à L. tropica, prêtant à confusion cliniquement avec des LC à L. major. PMID:25810796

  12. Non-viral delivery systems for CRISPR/Cas9-based genome editing: Challenges and opportunities.

    Science.gov (United States)

    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.

  13. Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes

    Science.gov (United States)

    Ma, Enbo; Harrington, Lucas B.; O’Connell, Mitchell R.; Zhou, Kaihong; Doudna, Jennifer A.

    2015-01-01

    Summary Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9–guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide-RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA, and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family. PMID:26545076

  14. 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.

  15. CRISPR/Cas9 Inhibits Multiple Steps of HIV-1 Infection.

    Science.gov (United States)

    Yin, Lijuan; Hu, Siqi; Mei, Shan; Sun, Hong; Xu, Fengwen; Li, Jian; Zhu, Weijun; Liu, Xiaoman; Zhao, Fei; Zhang, Di; Cen, Shan; Liang, Chen; Guo, Fei

    2018-05-09

    CRISPR/Cas9 is an adaptive immune system where bacteria and archaea have evolved to resist the invading viruses and plasmid DNA by creating site-specific double-strand breaks in DNA. This study tested this gene editing system in inhibiting human immunodeficiency virus type 1 (HIV-1) infection by targeting the viral long terminal repeat and the gene coding sequences. Strong inhibition of HIV-1 infection by Cas9/gRNA was observed, which resulted not only from insertions and deletions (indels) that were introduced into viral DNA due to Cas9 cleavage, but also from the marked decrease in the levels of the late viral DNA products and the integrated viral DNA. This latter defect might have reflected the degradation of viral DNA that has not been immediately repaired after Cas9 cleavage. It was further observed that Cas9, when solely located in the cytoplasm, inhibits HIV-1 as strongly as the nuclear Cas9, except that the cytoplasmic Cas9 does not act on the integrated HIV-1 DNA and thus cannot be used to excise the latent provirus. Together, the results suggest that Cas9/gRNA is able to target and edit HIV-1 DNA both in the cytoplasm and in the nucleus. The inhibitory effect of Cas9 on HIV-1 is attributed to both the indels in viral DNA and the reduction in the levels of viral DNA.

  16. 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

  17. Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR proteins.

    Science.gov (United States)

    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.

  18. Selected papers from the 2nd IEEEE Nordic Circuits and Systems Conference (NorCAS), 2016

    DEFF Research Database (Denmark)

    Sparsø, Jens

    2018-01-01

    This special issue includes selected papers from the 2nd IEEEE Nordic Circuits and Systems Conference (NorCAS), held in Linköping, Sweden, October 24-25, 2016. The IEEE NorCAS conference is the main circuits and systems event of the Nordic and Baltic countries representing both academia and the e......This special issue includes selected papers from the 2nd IEEEE Nordic Circuits and Systems Conference (NorCAS), held in Linköping, Sweden, October 24-25, 2016. The IEEE NorCAS conference is the main circuits and systems event of the Nordic and Baltic countries representing both academia...

  19. Diagnostic étiologique du diabète insipide central: à propos de 41 cas

    Science.gov (United States)

    Chaker, Fatma; Chihaoui, Melika; Yazidi, Meriem; Slimane, Hedia

    2016-01-01

    La survenue d'un syndrome polyuro-polydipsiqueavec des urines hypotoniques nécessite une stratégie diagnostique rigoureuse. Le but de cette étude était d’étudier les modalités de diagnostic du diabète insipide central. A travers une étude rétrospective de 41 cas de diabète insipide central(DIC) colligés au service d'Endocrinologie à l'hôpital de la Rabta de Tunis, allant de l'année 1990 à l'an 2013, nous avons relevé les circonstances de découverte du DIC, les anomalies du bilan anté-hypophysaire etde l'imagerie hypophysaire. Le DIC était post opératoire chez 20 patients. La diurèse moyenne de 24 heures était significativement plus élevée chez les patients ayant un DIC en dehors d'un contexte chirurgical. L’épreuve de restriction hydrique était concluante chez tous les patients qui en ont bénéficié. En dehors d'un contexte neurochirurgical, les causes infiltratives étaient retrouvées chez 6 patientset les causes tumorales chez 6 patients. Le DIC était associé à une selle turcique vide dans 1 cas et idiopathique chez 3 malades. L'imagerie par résonnance magnétique hypothalamo-hypophysaire et le bilan anté-hypophysaire sont systématiques en dehors d'un contexte de chirurgie hypophysaire et d'une polydipsie primaire évidente. PMID:27642481

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

    Science.gov (United States)

    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.

  1. 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.

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

    Science.gov (United States)

    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.

  3. [RS-1 enhanced the efficiency of CRISPR-Cas9 mediated knock-in of human lactoferrin].

    Science.gov (United States)

    Zhou, Wenjun; Guo, Rihong; Deng, Mingtian; Wang, Feng; Zhang, Yanli

    2017-08-25

    This study aims to knock out the goat β-lactoglobulin (BLG) gene using CRISPR-Cas9 system and knock in human lactoferrin (hLF) at the BLG locus, and further study the effect of RAD51 stimulatory compound (RS-1) on homologous recombination efficiency. First, we designed an sgRNA targeting the first exon of goat BLG gene and constructed a co-expression vector pCas9-sgBLG. This sgRNA vector was then transfected into goat ear fibroblasts (GEFs), and the target region was examined by T7EN1 assay and sequencing. Second, we constructed a targeting vector pBHA-hLF-NIE including NEO and EGFP genes based on BLG gene locus. This targeting vector together with pCas9-sgBLG expression vector was co-transfected into GEFs. Transfected cells were then treated with 0, 5, 10 and 20 μmol/L RS-1 for 72 h to analyse the EGFP expression efficiency. Next, we used 800 μg/mL G418 to screen G418-resistent cell clones, and studied hLF site-specific knock-in cell clones by PCR and sequencing. The editing efficiency of sgBLG was between 25% and 31%. The EGFP expression efficiency indicated that the gene knock-in efficiency was improved by RS-1 in a dose-dependent manner, which could reach 3.5-fold compared to the control group. The percentage of positive cells with hLF knock-in was increased to 32.61% when 10 μmol/L RS-1 was used. However, when the concentration of RS-1 increased to 20 μmol/L, the percentage of positive cells decreased to 22.22% and resulted in an increase of senescent cell clone number. These results suggested that hLF knock-in and BLG knock-out in GEFs were achieved by using CRISPR/Cas9 system, and optimum concentration of RS-1 could improve knock-in efficiency, which provides a reference for efficiently obtaining gene knock-in cells using CRISPR/Cas9 in the future.

  4. Analyse des facteurs prédictifs de malignité des goitres nodulaires : à propos de 500 cas

    Science.gov (United States)

    Bouaity, Brahim; Darouassi, Youssef; Chihani, Mehdi; Touati, Mohamed Mliha; Ammar, Haddou

    2016-01-01

    Les nodules thyroïdiens sont très fréquents et moins de 10% d'entre eux sont malin. Ils posent un véritable problème diagnostique et thérapeutique surtout par rapport à leur nature bénigne ou maligne. L’étude de certains facteurs cliniques et paracliniques de présomption de malignité permet de bien codifier la stratégie thérapeutique. Le but de ce travail est d’étudier les facteurs prédictifs de malignité des goitres nodulaires et comparer nos résultats à ceux de la littérature. Il s'agit d'une étude rétrospective à propos de 500 cas de goitres nodulaires opérés au service d'Oto-rhino-laryngologie (ORL) et Chirurgie cervico-faciale (CCF) de l'hôpital militaire Avicenne de Marrakech entre 2006 et 2012. Le pourcentage de cancers a été de 6,8%. L’âge moyen de nos patients était de 46 ans, avec une sex-ratio de 5 (F/H). A la palpation cervicale; le caractère dure du nodule a été constaté dans 94,4% des cas de cancer, avec des limites irrégulières dans 64,70% des cas de cancer. Trois nodules étaient fixes et ils étaient tous malins. Les adénopathies cervicales ont été constatées chez 8 malades dont 7 présentaient des cancers. A l’échographie, 61,8% des nodules malins présentaient un aspect hypoéchogène, avec des contours flous dans 88,24% des cas. La vascularisation intra nodulaire était présente dans 35,3% de ces cas des cancers avec des microcalcifications chez 55,9% d'entre eux. Le halo hypoéchogene périnodulaire était incomplet dans 73,5% des cas de cancer. Nos patients étaient en euthyroïdie dans 84,6% des cas. Les facteurs prédictifs de malignité d'un goitre nodulaire, étaient donc dans notre étude d'abord cliniques: l’âge supérieur à 60 ans, la consistance dure du nodule, sa fixité, son caractère irrégulier et mal limité à la palpation, ainsi que la présence d'adénopathie(s) cervicale(s) à l'examen; et échographiques: le caractère hypoéchogène, les limites floues, la présence de

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

    Science.gov (United States)

    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.

  6. Generation of a Nrf2 homozygous knockout human embryonic stem cell line using CRISPR/Cas9

    Directory of Open Access Journals (Sweden)

    So-Jung Kim

    2017-03-01

    Full Text Available Nuclear factor erythroid 2-related factor 2 (NFE2L2 or Nrf2 is a well-known transcription factor that regulates the expression of a large number of anti-oxidant genes in mammalian cells (J.H. Kim et al., 2014. Here, we generated a homozygous Nrf2 knockout human embryonic stem cell (hESC line, H9Nrf2KO-A13, using the CRISPR/Cas9 genome editing method. The Nrf2 homozygous knockout H9 cell line maintains pluripotency, differentiation potential into three germ layers, and a normal karyotype.

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

    Science.gov (United States)

    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

  8. Student Perceptions of Academic Service Learning: Using Mixed Content Analysis to Examine the Effectiveness of the International Baccalaureate Creativity, Action, Service Programme

    Science.gov (United States)

    Hatziconstantis, Christos; Kolympari, Tania

    2016-01-01

    The International Baccalaureate Diploma Programme for secondary education students requires the successful completion of the Creativity, Action, Service (CAS) component (more recently renamed Creativity, Activity, Service) which is based on the philosophy of experiential learning and Academic Service Learning. In this article, the technique of…

  9. 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

  10. CRISPR-Cas: evolution of an RNA-based adaptive immunity system in prokaryotes.

    Science.gov (United States)

    Koonin, Eugene V; Makarova, Kira S

    2013-05-01

    The CRISPR-Cas (clustered regularly interspaced short palindromic repeats, CRISPR-associated genes) is an adaptive immunity system in bacteria and archaea that functions via a distinct self-non-self recognition mechanism that is partially analogous to the mechanism of eukaryotic RNA interference (RNAi). The CRISPR-Cas system incorporates fragments of virus or plasmid DNA into the CRISPR repeat cassettes and employs the processed transcripts of these spacers as guide RNAs to cleave the cognate foreign DNA or RNA. The Cas proteins, however, are not homologous to the proteins involved in RNAi and comprise numerous, highly diverged families. The majority of the Cas proteins contain diverse variants of the RNA recognition motif (RRM), a widespread RNA-binding domain. Despite the fast evolution that is typical of the cas genes, the presence of diverse versions of the RRM in most Cas proteins provides for a simple scenario for the evolution of the three distinct types of CRISPR-cas systems. In addition to several proteins that are directly implicated in the immune response, the cas genes encode a variety of proteins that are homologous to prokaryotic toxins that typically possess nuclease activity. The predicted toxins associated with CRISPR-Cas systems include the essential Cas2 protein, proteins of COG1517 that, in addition to a ligand-binding domain and a helix-turn-helix domain, typically contain different nuclease domains and several other predicted nucleases. The tight association of the CRISPR-Cas immunity systems with predicted toxins that, upon activation, would induce dormancy or cell death suggests that adaptive immunity and dormancy/suicide response are functionally coupled. Such coupling could manifest in the persistence state being induced and potentially providing conditions for more effective action of the immune system or in cell death being triggered when immunity fails.

  11. Mobile Genetic Elements and Evolution of CRISPR-Cas Systems: All the Way There and Back

    Science.gov (United States)

    Makarova, Kira S.

    2017-01-01

    Abstract The Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-CRISPR-associated proteins (Cas) systems of bacterial and archaeal adaptive immunity show multifaceted evolutionary relationships with at least five classes of mobile genetic elements (MGE). First, the adaptation module of CRISPR-Cas that is responsible for the formation of the immune memory apparently evolved from a Casposon, a self-synthesizing transposon that employs the Cas1 protein as the integrase and might have brought additional cas genes to the emerging immunity loci. Second, a large subset of type III CRISPR-Cas systems recruited a reverse transcriptase from a Group II intron, providing for spacer acquisition from RNA. Third, effector nucleases of Class 2 CRISPR-Cas systems that are responsible for the recognition and cleavage of the target DNA were derived from transposon-encoded TnpB nucleases, most likely, on several independent occasions. Fourth, accessory nucleases in some variants of types I and III toxin and type VI effectors RNases appear to be ultimately derived from toxin nucleases of microbial toxin–antitoxin modules. Fifth, the opposite direction of evolution is manifested in the recruitment of CRISPR-Cas systems by a distinct family of Tn7-like transposons that probably exploit the capacity of CRISPR-Cas to recognize unique DNA sites to facilitate transposition as well as by bacteriophages that employ them to cope with host defense. Additionally, individual Cas proteins, such as the Cas4 nuclease, were recruited by bacteriophages and transposons. The two-sided evolutionary connection between CRISPR-Cas and MGE fits the “guns for hire” paradigm whereby homologous enzymatic machineries, in particular nucleases, are shuttled between MGE and defense systems and are used alternately as means of offense or defense. PMID:28985291

  12. Achieving a milestone: AMOR is now archived in Portico and indexed in CAS

    Directory of Open Access Journals (Sweden)

    R.N Sugitha Nadarajah

    2016-10-01

    Full Text Available The journal of Advances in Modern Oncology Research (AMOR is proud to announce its partnership with Portico, a leading digital preservation service provider, to ensure the long term accessibility of its published contents. In addition, it is now indexed by the gold standard of chemical information database Chemical Abstracts Services (CAS after publishing only six bimonthly issues, dating back to October 2015. AMOR’s Editor-in-Chief Dr. Omar Abdel-Rahman proudly shares: “Our vision is to be far more than just a journal, we want to be a platform that publishes high quality cancer research contents from all over the world. I think we are progressing, at an acceptable pace, in that direction."The Portico archive is a “centralized repository of tens of thousands of e-journals, e-books, and other electronic content, replicated to ensure security,” according to the registry organization. “Content comes into the archive under formal preservation agreements with publishers. Content providers submit source files to Portico, and we repackage these source files into an archival format and provide long-term archival management and format migration as needed. Our approach is driven by our commitment to meeting clear preservation goals,” it states. AMOR’s Managing Editor Dr. TS Jong, when asked to comment about the impact of this development, adds: “AMOR is committed towards meeting the highest international publication standards and as such, has a clear archiving and indexing roadmap to sustain our growth. The journal acknowledges the importance of ensuring the continuous accessibility of our published articles in multiple repositories. Therefore, we are delighted with the recent partnership between AMOR and Portico, a well-known third-party repository service provider, to safeguard the long-term availability of our contents.”Archiving AMOR’s articles within Portico ensures that the materials published are always available for access. In

  13. CONFUSION WITH TELEPHONE NUMBERS

    CERN Multimedia

    Telecom Service

    2002-01-01

    he area code is now required for all telephone calls within Switzerland. Unfortunately this is causing some confusion. CERN has received complaints that incoming calls intended for CERN mobile phones are being directed to private subscribers. This is caused by mistakenly dialing the WRONG code (e.g. 022) in front of the mobile number. In order to avoid these problems, please inform your correspondents that the correct numbers are: 079 201 XXXX from Switzerland; 0041 79 201 XXXX from other countries. Telecom Service

  14. CONFUSION WITH TELEPHONE NUMBERS

    CERN Multimedia

    Telecom Service

    2002-01-01

    The area code is now required for all telephone calls within Switzerland. Unfortunately this is causing some confusion. CERN has received complaints that incoming calls intended for CERN mobile phones are being directed to private subscribers. This is caused by mistakenly dialing the WRONG code (e.g. 022) in front of the mobile number. In order to avoid these problems, please inform your correspondents that the correct numbers are: 079 201 XXXX from Switzerland; 0041 79 201 XXXX from other countries. Telecom Service  

  15. CRISPR-Cas9 directed knock-out of a constitutively expressed gene using lance array nanoinjection.

    Science.gov (United States)

    Sessions, John W; Skousen, Craig S; Price, Kevin D; Hanks, Brad W; Hope, Sandra; Alder, Jonathan K; Jensen, Brian D

    2016-01-01

    CRISPR-Cas9 genome editing and labeling has emerged as an important tool in biologic research, particularly in regards to potential transgenic and gene therapy applications. Delivery of CRISPR-Cas9 plasmids to target cells is typically done by non-viral methods (chemical, physical, and/or electrical), which are limited by low transfection efficiencies or with viral vectors, which are limited by safety and restricted volume size. In this work, a non-viral transfection technology, named lance array nanoinjection (LAN), utilizes a microfabricated silicon chip to physically and electrically deliver genetic material to large numbers of target cells. To demonstrate its utility, we used the CRISPR-Cas9 system to edit the genome of isogenic cells. Two variables related to the LAN process were tested which include the magnitude of current used during plasmid attraction to the silicon lance array (1.5, 4.5, or 6.0 mA) and the number of times cells were injected (one or three times). Results indicate that most successful genome editing occurred after injecting three times at a current control setting of 4.5 mA, reaching a median level of 93.77 % modification. Furthermore, we found that genome editing using LAN follows a non-linear injection-dose response, meaning samples injected three times had modification rates as high as nearly 12 times analogously treated single injected samples. These findings demonstrate the LAN's ability to deliver genetic material to cells and indicate that successful alteration of the genome is influenced by a serial injection method as well as the electrical current settings.

  16. La mediazione familiare nei casi di affido dei figli/e e violenza domestica: contesto legale, pratiche dei servizi ed esperienze delle donne in Italia / Family mediation in child custody cases and domestic violence: legal context, logic of services and women's experiences in Italy / La médiation familiale dans les cas de garde d’enfants et la violence conjugale : le contexte juridique, les pratiques au sein des services et les expériences des femmes en Italie

    Directory of Open Access Journals (Sweden)

    Mariachiara Feresin

    2017-07-01

    Full Text Available L’applicabilità della mediazione familiare in contesto di violenza domestica (VD è oggetto di discussione. Scopo della ricerca è esplorare il ruolo della mediazione familiare nella gestione degli affidi dei figli in situazione di VD, analizzando le esperienze, conoscenze e significati di differenti attori sociali, quali avvocati, assistenti sociali e donne separate con figli, vittime di VD, e la documentazione inerente. I risultati mostrano che la VD viene occultata durante la mediazione. I professionisti spesso ignorano la VD e di conseguenza applicano la mediazione; ex-coniugi e genitori vengono presentati come distinti; i pattern di potere e controllo agiti dal partner violento durante la relazione continuano in queste occasioni. La mediazione, che dovrebbe essere centrata sul miglior interesse del bambino, si focalizza sul miglior interesse dei padri. I professionisti non conoscono la Convenzione di Istanbul. La sicurezza di donne e bambini/e viene messa a rischio. Le recours à la médiation familiale dans le domaine de la violence conjugale (VC fait l’objet de débats. Cette recherche a pour but d’examiner le rôle de la médiation familiale dans les cas de garde d’enfants en situation de VC, analysant les expériences, les connaissances, les valeurs de différents acteurs sociaux (par exemple, avocats, travailleurs sociaux, femmes séparées avec enfants, victimes de VC ainsi que des documents ad hoc. Les résultats montrent que la VC est dissimulée pendant la médiation. Les professionnels souvent ignorent la VC et par conséquent utilisent la médiation ; ex-conjoints et parents sont par ailleurs présentés sous la forme de deux entités distinctes ; les modèles de pouvoir et de contrôle appliqués par le conjoint violent dans la vie familiale continuent d’être utilisés durant ces occasions. La médiation, qui devrait protéger avant tout l'intérêt de l'enfant, s’adresse au contraire à l’intérêt des p

  17. Engineering resistance against Tomato yellow leaf curl virus via the CRISPR/Cas9 system in tomato

    KAUST Repository

    Mahfouz, Magdy M.; Tashkandi, Manal; Ali, Zahir; Aljedaani, Fatimah R.; Shami, Ashwag

    2017-01-01

    CRISPR/Cas systems confer molecular immunity against phages and conjugative plasmids in prokaryotes. Recently, CRISPR/Cas9 systems have been used to confer interference against eukaryotic viruses. Here, we engineered Nicotiana benthamiana and tomato

  18. Baculoviral delivery of CRISPR/Cas9 facilitates efficient genome editing in human cells

    NARCIS (Netherlands)

    Hindriksen, Sanne; Bramer, Arne J; Truong, My Anh; Vromans, Martijn J M; Post, Jasmin B; Verlaan-Klink, Ingrid; Snippert, Hugo J; Lens, Susanne M A; Hadders, Michael A

    2017-01-01

    The CRISPR/Cas9 system is a highly effective tool for genome editing. Key to robust genome editing is the efficient delivery of the CRISPR/Cas9 machinery. Viral delivery systems are efficient vehicles for the transduction of foreign genes but commonly used viral vectors suffer from a limited

  19. 76 FR 79545 - Cost Accounting Standards: Change to the CAS Applicability Threshold for the Inflation Adjustment...

    Science.gov (United States)

    2011-12-22

    ... Cost Accounting Standards: Change to the CAS Applicability Threshold for the Inflation Adjustment to... Federal Procurement Policy, Cost Accounting Standards Board. ACTION: Final rule. SUMMARY: The Office of Federal Procurement Policy (OFPP), Cost Accounting Standards (CAS) Board (Board), has adopted, without...

  20. Computational Neural Modeling of Speech Motor Control in Childhood Apraxia of Speech (CAS)

    Science.gov (United States)

    Terband, Hayo; Maassen, Ben; Guenther, Frank H.; Brumberg, Jonathan

    2009-01-01

    Purpose: Childhood apraxia of speech (CAS) has been associated with a wide variety of diagnostic descriptions and has been shown to involve different symptoms during successive stages of development. In the present study, the authors attempted to associate the symptoms of CAS in a particular developmental stage with particular…

  1. Phonological Awareness and Early Reading Development in Childhood Apraxia of Speech (CAS)

    Science.gov (United States)

    McNeill, B. C.; Gillon, G. T.; Dodd, B.

    2009-01-01

    Background: Childhood apraxia of speech (CAS) is associated with phonological awareness, reading, and spelling deficits. Comparing literacy skills in CAS with other developmental speech disorders is critical for understanding the complexity of the disorder. Aims: This study compared the phonological awareness and reading development of children…

  2. Computational neural modeling of speech motor control in childhood apraxia of speech (CAS).

    NARCIS (Netherlands)

    Terband, H.R.; Maassen, B.A.M.; Guenther, F.H.; Brumberg, J.

    2009-01-01

    PURPOSE: Childhood apraxia of speech (CAS) has been associated with a wide variety of diagnostic descriptions and has been shown to involve different symptoms during successive stages of development. In the present study, the authors attempted to associate the symptoms of CAS in a particular

  3. An Undergraduate Laboratory Class Using CRISPR/Cas9 Technology to Mutate Drosophila Genes

    Science.gov (United States)

    Adame, Vanesa; Chapapas, Holly; Cisneros, Marilyn; Deaton, Carol; Deichmann, Sophia; Gadek, Chauncey; Lovato, TyAnna L.; Chechenova, Maria B.; Guerin, Paul; Cripps, Richard M.

    2016-01-01

    CRISPR/Cas9 genome editing technology is used in the manipulation of genome sequences and gene expression. Because of the ease and rapidity with which genes can be mutated using CRISPR/Cas9, we sought to determine if a single-semester undergraduate class could be successfully taught, wherein students isolate mutants for specific genes using…

  4. Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering

    KAUST Repository

    Aouida, Mustapha; Eid, Ayman; Ali, Zahir; Cradick, Thomas; Lee, Ciaran; Deshmukh, Harshavardhan; Atef, Ahmed; Abu Samra, Dina Bashir Kamil; Gadhoum, Samah Zeineb; Merzaban, Jasmeen; Bao, Gang; Mahfouz, Magdy M.

    2015-01-01

    The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15–39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.

  5. p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells.

    Science.gov (United States)

    Ihry, Robert J; Worringer, Kathleen A; Salick, Max R; Frias, Elizabeth; Ho, Daniel; Theriault, Kraig; Kommineni, Sravya; Chen, Julie; Sondey, Marie; Ye, Chaoyang; Randhawa, Ranjit; Kulkarni, Tripti; Yang, Zinger; McAllister, Gregory; Russ, Carsten; Reece-Hoyes, John; Forrester, William; Hoffman, Gregory R; Dolmetsch, Ricardo; Kaykas, Ajamete

    2018-06-11

    CRISPR/Cas9 has revolutionized our ability to engineer genomes and conduct genome-wide screens in human cells 1-3 . Whereas some cell types are amenable to genome engineering, genomes of human pluripotent stem cells (hPSCs) have been difficult to engineer, with reduced efficiencies relative to tumour cell lines or mouse embryonic stem cells 3-13 . Here, using hPSC lines with stable integration of Cas9 or transient delivery of Cas9-ribonucleoproteins (RNPs), we achieved an average insertion or deletion (indel) efficiency greater than 80%. This high efficiency of indel generation revealed that double-strand breaks (DSBs) induced by Cas9 are toxic and kill most hPSCs. In previous studies, the toxicity of Cas9 in hPSCs was less apparent because of low transfection efficiency and subsequently low DSB induction 3 . The toxic response to DSBs was P53/TP53-dependent, such that the efficiency of precise genome engineering in hPSCs with a wild-type P53 gene was severely reduced. Our results indicate that Cas9 toxicity creates an obstacle to the high-throughput use of CRISPR/Cas9 for genome engineering and screening in hPSCs. Moreover, as hPSCs can acquire P53 mutations 14 , cell replacement therapies using CRISPR/Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function.

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

    Science.gov (United States)

    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.

  7. Single step production of Cas9 mRNA for zygote injection.

    Science.gov (United States)

    Redel, Bethany K; Beaton, Benjamin P; Spate, Lee D; Benne, Joshua A; Murphy, Stephanie L; O'Gorman, Chad W; Spate, Anna M; Prather, Randall S; Wells, Kevin D

    2018-03-01

    Production of Cas9 mRNA in vitro typically requires the addition of a 5´ cap and 3´ polyadenylation. A plasmid was constructed that harbored the T7 promoter followed by the EMCV IRES and a Cas9 coding region. We hypothesized that the use of the metastasis associated lung adenocarcinoma transcript 1 (Malat1) triplex structure downstream of an IRES/Cas9 expression cassette would make polyadenylation of in vitro produced mRNA unnecessary. A sequence from the mMalat1 gene was cloned downstream of the IRES/Cas9 cassette described above. An mRNA concentration curve was constructed with either commercially available Cas9 mRNA or the IRES/ Cas9/triplex, by injection into porcine zygotes. Blastocysts were genotyped to determine if differences existed in the percent of embryos modified. The concentration curve identified differences due to concentration and RNA type injected. Single step production of Cas9 mRNA provides an alternative source of Cas9 for use in zygote injections.

  8. Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryos

    Science.gov (United States)

    Tu, Zhuchi; Yang, Weili; Yan, Sen; Yin, An; Gao, Jinquan; Liu, Xudong; Zheng, Yinghui; Zheng, Jiezhao; Li, Zhujun; Yang, Su; Li, Shihua; Guo, Xiangyu; Li, Xiao-Jiang

    2017-01-01

    CRISPR-Cas9 is a powerful new tool for genome editing, but this technique creates mosaic mutations that affect the efficiency and precision of its ability to edit the genome. Reducing mosaic mutations is particularly important for gene therapy and precision genome editing. Although the mechanisms underlying the CRSIPR/Cas9-mediated mosaic mutations remain elusive, the prolonged expression and activity of Cas9 in embryos could contribute to mosaicism in DNA mutations. Here we report that tagging Cas9 with ubiquitin-proteasomal degradation signals can facilitate the degradation of Cas9 in non-human primate embryos. Using embryo-splitting approach, we found that shortening the half-life of Cas9 in fertilized zygotes reduces mosaic mutations and increases its ability to modify genomes in non-human primate embryos. Also, injection of modified Cas9 in one-cell embryos leads to live monkeys with the targeted gene modifications. Our findings suggest that modifying Cas9 activity can be an effective strategy to enhance precision genome editing. PMID:28155910

  9. Mutagenesis of FAD2 genes in peanut with CRISPR/Cas9

    Science.gov (United States)

    The CRISPR/Cas9 system is known for its precise and efficient gene-editing of a targeted region in a variety of organisms including plants. We targeted FAD2 gene region to perform CRISPR/Cas9 gene-editing in peanut. The FAD2 gene encodes fatty acid desaturase which catalyzes the conversion of oleic ...

  10. CRISPR/Cas systems: new players in gene regulation and bacterial physiology

    Directory of Open Access Journals (Sweden)

    David eWeiss

    2014-04-01

    Full Text Available CRISPR-Cas systems are bacterial defenses against foreign nucleic acids derived from bacteriophages, plasmids or other sources. These systems are targeted in an RNA-dependent, sequence-specific manner, and are also adaptive, providing protection against previously encountered foreign elements. In addition to their canonical function in defense against foreign nucleic acid, their roles in various aspects of bacterial physiology are now being uncovered. We recently revealed a role for a Cas9-based Type II CRISPR-Cas system in the control of endogenous gene expression, a novel form of prokaryotic gene regulation. Cas9 functions in association with two small RNAs to target and alter the stability of an endogenous transcript encoding a bacterial lipoprotein (BLP. Since BLPs are recognized by the host innate immune protein Toll-like Receptor 2 (TLR2, CRISPR-Cas-mediated repression of BLP expression facilitates evasion of TLR2 by the intracellular bacterial pathogen Francisella novicida, and is essential for its virulence. Here we describe the Cas9 regulatory system in detail, as well as data on its role in controlling virulence traits of Neisseria meningitidis and Campylobacter jejuni. We also discuss potential roles of CRISPR-Cas systems in the response to envelope stress and other aspects of bacterial physiology. Since ~45% of bacteria and ~83% of Archaea encode these machineries, the newly appreciated regulatory functions of CRISPR-Cas systems are likely to play broad roles in controlling the pathogenesis and physiology of diverse prokaryotes.

  11. Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering

    KAUST Repository

    Aouida, Mustapha

    2015-07-30

    The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15–39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.

  12. Using local chromatin structure to improve CRISPR/Cas9 efficiency in zebrafish.

    Science.gov (United States)

    Chen, Yunru; Zeng, Shiyang; Hu, Ruikun; Wang, Xiangxiu; Huang, Weilai; Liu, Jiangfang; Wang, Luying; Liu, Guifen; Cao, Ying; Zhang, Yong

    2017-01-01

    Although the CRISPR/Cas9 has been successfully applied in zebrafish, considerable variations in efficiency have been observed for different gRNAs. The workload and cost of zebrafish mutant screening is largely dependent on the mutation rate of injected embryos; therefore, selecting more effective gRNAs is especially important for zebrafish mutant construction. Besides the sequence features, local chromatin structures may have effects on CRISPR/Cas9 efficiency, which remain largely unexplored. In the only related study in zebrafish, nucleosome organization was not found to have an effect on CRISPR/Cas9 efficiency, which is inconsistent with recent studies in vitro and in mammalian cell lines. To understand the effects of local chromatin structure on CRISPR/Cas9 efficiency in zebrafish, we first determined that CRISPR/Cas9 introduced genome editing mainly before the dome stage. Based on this observation, we reanalyzed our published nucleosome organization profiles and generated chromatin accessibility profiles in the 256-cell and dome stages using ATAC-seq technology. Our study demonstrated that chromatin accessibility showed positive correlation with CRISPR/Cas9 efficiency, but we did not observe a clear correlation between nucleosome organization and CRISPR/Cas9 efficiency. We constructed an online database for zebrafish gRNA selection based on local chromatin structure features that could prove beneficial to zebrafish homozygous mutant construction via CRISPR/Cas9.

  13. Engineering Plants for Geminivirus Resistance with CRISPR/Cas9 System

    KAUST Repository

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

    2016-01-01

    The CRISPR/Cas9 system is an efficient genome-editing platform for diverse eukaryotic species, including plants. Recent work harnessed CRISPR/Cas9 technology to engineer resistance to geminiviruses. Here, we discuss opportunities, emerging developments, and potential pitfalls for using this technology to engineer resistance against single and multiple geminivirus infections in plants.

  14. Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice.

    Science.gov (United States)

    Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

    2015-08-01

    The CRISPR/Cas9 system is an efficient tool used for genome editing in a variety of organisms. Despite several recent reports of successful targeted mutagenesis using the CRISPR/Cas9 system in plants, in each case the target gene of interest, the Cas9 expression system and guide-RNA (gRNA) used, and the tissues used for transformation and subsequent mutagenesis differed, hence the reported frequencies of targeted mutagenesis cannot be compared directly. Here, we evaluated mutation frequency in rice using different Cas9 and/or gRNA expression cassettes under standardized experimental conditions. We introduced Cas9 and gRNA expression cassettes separately or sequentially into rice calli, and assessed the frequency of mutagenesis at the same endogenous targeted sequences. Mutation frequencies differed significantly depending on the Cas9 expression cassette used. In addition, a gRNA driven by the OsU6 promoter was superior to one driven by the OsU3 promoter. Using an all-in-one expression vector harboring the best combined Cas9/gRNA expression cassette resulted in a much improved frequency of targeted mutagenesis in rice calli, and bi-allelic mutant plants were produced in the T0 generation. The approach presented here could be adapted to optimize the construction of Cas9/gRNA cassettes for genome editing in a variety of plants.

  15. Repetitive DNA Reeling by the Cascade-Cas3 Complex in Nucleotide Unwinding Steps

    NARCIS (Netherlands)

    Loeff, Luuk; Brouns, Stan J.J.; Joo, Chirlmin

    2018-01-01

    CRISPR-Cas provides RNA-guided adaptive immunity against invading genetic elements. Interference in type I systems relies on the RNA-guided Cascade complex for target DNA recognition and the Cas3 helicase/nuclease protein for target degradation. Even though the biochemistry of CRISPR interference

  16. CAS Panel Proposes Priorities for Earth Science in Next Two Decades

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    @@ CAS member Zhao Zhongxian, director of Working Committee on Consultation and Evaluation of the CAS Academic Divisions (CASAD),has announced that the Academic Division of Earth Sciences has drafted a consultative report on planning and strategic studies of the mid- and long-term development for earth sciences in China.

  17. 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

  18. Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems

    NARCIS (Netherlands)

    Mohanraju, Prarthana; Makarova, Kira S.; Zetsche, Bernd; Zhang, Feng; Koonin, Eugene V.; Oost, van der John

    2016-01-01

    Adaptive immunity had been long thought of as an exclusive feature of animals. However, the discovery of the CRISPR-Cas defense system, present in almost half of prokaryotic genomes, proves otherwise. Because of the everlasting parasite-host arms race, CRISPR-Cas has rapidly evolved through

  19. 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.

  20. Optimization of genome engineering approaches with the CRISPR/Cas9 system

    DEFF Research Database (Denmark)

    Li, Kai; Wang, Gang; Andersen, Troels

    2014-01-01

    Designer nucleases such as TALENS and Cas9 have opened new opportunities to scarlessly edit the mammalian genome. Here we explored several parameters that influence Cas9-mediated scarless genome editing efficiency in murine embryonic stem cells. Optimization of transfection conditions and enrichi...

  1. 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

  2. Engineering Plants for Geminivirus Resistance with CRISPR/Cas9 System

    KAUST Repository

    Zaidi, Syed Shan-e-Ali

    2016-02-14

    The CRISPR/Cas9 system is an efficient genome-editing platform for diverse eukaryotic species, including plants. Recent work harnessed CRISPR/Cas9 technology to engineer resistance to geminiviruses. Here, we discuss opportunities, emerging developments, and potential pitfalls for using this technology to engineer resistance against single and multiple geminivirus infections in plants.

  3. 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

  4. Structural Plasticity of PAM Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9.

    Science.gov (United States)

    Anders, Carolin; Bargsten, Katja; Jinek, Martin

    2016-03-17

    The RNA-guided endonuclease Cas9 from Streptococcus pyogenes (SpCas9) forms the core of a powerful genome editing technology. DNA cleavage by SpCas9 is dependent on the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) in the target DNA, restricting the choice of targetable sequences. To address this limitation, artificial SpCas9 variants with altered PAM specificities have recently been developed. Here we report crystal structures of the VQR, EQR, and VRER SpCas9 variants bound to target DNAs containing their preferred PAM sequences. The structures reveal that the non-canonical PAMs are recognized by an induced fit mechanism. Besides mediating sequence-specific base recognition, the amino acid substitutions introduced in the SpCas9 variants facilitate conformational remodeling of the PAM region of the bound DNA. Guided by the structural data, we engineered a SpCas9 variant that specifically recognizes NAAG PAMs. Taken together, these studies inform further development of Cas9-based genome editing tools. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. 78 FR 40665 - Cost Accounting Standards: CAS 413 Pension Adjustments for Extraordinary Events

    Science.gov (United States)

    2013-07-08

    ... Accounting Standards: CAS 413 Pension Adjustments for Extraordinary Events AGENCY: Cost Accounting Standards...: The Office of Federal Procurement Policy (OFPP), Cost Accounting Standards (CAS) Board, is conducting... Extraordinary Events. This is the first step in a four- step process that may result in a final rule. As part of...

  6. Poison control center - emergency number

    Science.gov (United States)

    For a POISON EMERGENCY call: 1-800-222-1222 ANYWHERE IN THE UNITED STATES This national hotline number will let you ... is a free and confidential service. All local poison control centers in the United States use this ...

  7. Auditing the Functional Part of the CAS Software

    Directory of Open Access Journals (Sweden)

    Adamyk Oksana V.

    2017-11-01

    Full Text Available The article is aimed at determining the order and methodology of auditing the functional component of the software for an computer accounting system (CAS. It has been found that software auditing should be performed separately for each of its components. The components of the functional part of the CAS software are the database management system (DBMS and the application software supporting the accountance automation. For auditing of the first component part are used such techniques as general evaluation, subject check of the embedded algorithms of information processing. Auditing the client software algorithms is carried out by means of the control data method, which is reduced to such procedures as creation of another database of test data with imaginary objects and its processing by the client program, as well as introduction in a copy of the real database of imaginary objects (employees, creditors, material values and the formation of reporting. Not only the current methods of calculation or evaluation of accounting objects, but all of the software, are subject to mandatory verification. This will avoid errors if the enterprise accounting policy changes.

  8. The linear collider alignment and survey (LiCAS) project

    International Nuclear Information System (INIS)

    Bingham, Richard; Botcherby, Edward; Coe, Paul; Grzelak, Grzegorz; Mitra, Ankush; Reichold, Armin; Prenting, Johannes

    2003-01-01

    For the next generation of Linear Colliders (LC) the precision alignment of accelerator components will be critical. The DESY applied geodesy group has developed the concept of an automated 'survey train'. The train runs along the accelerator wall measuring the 3D position of a set of equispaced reference markers. This reference structure is then used to align the accelerator components. The LiCAS group is developing a measurement system for the survey train. It will use a combination of Laser Straightness Monitors (SM) and Frequency Scanning Interferometry (FSI). FSI is an interferometric length measurement technique originally developed for the online alignment of the ATLAS Inner Detector. This novel combination of optical techniques is expected to overcome the limitations of traditional open air survey. The authors describe the LiCAS project, the measurement systems and their integration into the survey train. The technical parameters and constraints will be mentioned. There will also be brief discussion of the second phase of the project to allow on-line monitoring of the LC alignment. (author)

  9. CAS-NETL-PNNL CEP Program Final Report

    Energy Technology Data Exchange (ETDEWEB)

    King, David L.; Spies, Kurt A.; Rainbolt, James E.; Zhang, Keling

    2014-03-31

    This collaborative joint research project is in the area of advanced gasification and conversion, within the CAS-NETL-PNNL Memorandum of Understanding. The goal is the development and testing of an integrated warm syngas cleanup process. This effort is focused on an advanced, integrated system for capture and removal of alkali, sulfur, PH3, AsH3, chloride, and CO2, leading to a future process demonstration at a CAS gasification facility. Syngas produced by gasification can be used for production of fuels (Fischer-Tropsch, SNG, mixed alcohols), chemicals (MeOH, NH3), and hydrogen for fuel cells and IGCC. To employ this syngas, especially for synthesis reactions, contained impurities must be removed to sub-ppmv levels [1]. Commercially available approaches to remove contaminant species suffer from inefficiencies, employing solvents at ambient or lower temperature along with backup sacrificial sorbents, whereas syngas utilization occurs at higher temperatures. The efficiency and economics syngas utilization can be significantly improved if all the contaminants and CO2 are removed at temperatures higher than the chemical synthesis reaction temperatures (> 250 °C) [2].

  10. Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies

    DEFF Research Database (Denmark)

    Damgaard Jensen, Emil; Ferreira, Raphael; Jakociunas, Tadas

    2017-01-01

    on developing synthetic biology tools for orthogonal control of transcription. Most recently, the nuclease-deficient Cas9 (dCas9) has emerged as a flexible tool for controlling activation and repression of target genes, by the simple RNA-guided positioning of dCas9 in the vicinity of the target gene...... transcription start site. In this study we compared two different systems of dCas9-mediated transcriptional reprogramming, and applied them to genes controlling two biosynthetic pathways for biobased production of isoprenoids and triacylglycerols (TAGs) in baker's yeast Saccharomyces cerevisiae. By testing 101...... production and increases in TAG. Taken together, we show similar performance for a constitutive and an inducible dCas9 approach, and identify multiplex gRNA designs that can significantly perturb isoprenoid production and TAG profiles in yeast without editing the genomic context of the target genes. We also...

  11. 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

  12. 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.

  13. CRISPR/Cas9-mediated target validation of the Splicing Inhibitor Pladienolide B

    KAUST Repository

    Aouida, Mustapha; Eid, Ayman; Mahfouz, Magdy M.

    2016-01-01

    CRISPR/Cas9 system confers molecular immunity in archeal and bacterial species against invading foreign nucleic acids. CRISPR/Cas9 system is used for genome engineering applications across diverse eukaryotic species. In this study, we demonstrate the utility of the CRISPR/Cas9 genome engineering system for drug target validation in human cells. Pladienolide B is a natural macrolide with antitumor activities mediated through the inhibition of pre-mRNA splicing. To validate the spliceosomal target of Pladienolide B, we employed the CRSIPR/Cas9 system to introduce targeted mutations in the subunits of the SF3B complex in the HEK293T cells. Our data reveal that targeted mutagenesis of the SF3b1 subunit exhibited higher levels of resistance to Pladienolide B. Therefore, our data validate the spliceosomal target of Pladienolide B and provide a proof of concept on using the CRISPR/Cas9 system for drug target identification and validation.

  14. CAS algorithm-based optimum design of PID controller in AVR system

    International Nuclear Information System (INIS)

    Zhu Hui; Li Lixiang; Zhao Ying; Guo Yu; Yang Yixian

    2009-01-01

    This paper presents a novel design method for determining the optimal PID controller parameters of an automatic voltage regulator (AVR) system using the chaotic ant swarm (CAS) algorithm. In the tuning process of parameters, the CAS algorithm is iterated to give the optimal parameters of the PID controller based on the fitness theory, where the position vector of each ant in the CAS algorithm corresponds to the parameter vector of the PID controller. The proposed CAS-PID controllers can ensure better control system performance with respect to the reference input in comparison with GA-PID controllers. Numerical simulations are provided to verify the effectiveness and feasibility of PID controller based on CAS algorithm.

  15. CRISPR/Cas9-mediated target validation of the Splicing Inhibitor Pladienolide B

    KAUST Repository

    Aouida, Mustapha

    2016-02-24

    CRISPR/Cas9 system confers molecular immunity in archeal and bacterial species against invading foreign nucleic acids. CRISPR/Cas9 system is used for genome engineering applications across diverse eukaryotic species. In this study, we demonstrate the utility of the CRISPR/Cas9 genome engineering system for drug target validation in human cells. Pladienolide B is a natural macrolide with antitumor activities mediated through the inhibition of pre-mRNA splicing. To validate the spliceosomal target of Pladienolide B, we employed the CRSIPR/Cas9 system to introduce targeted mutations in the subunits of the SF3B complex in the HEK293T cells. Our data reveal that targeted mutagenesis of the SF3b1 subunit exhibited higher levels of resistance to Pladienolide B. Therefore, our data validate the spliceosomal target of Pladienolide B and provide a proof of concept on using the CRISPR/Cas9 system for drug target identification and validation.

  16. Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum.

    Science.gov (United States)

    Liu, Jiao; Wang, Yu; Lu, Yujiao; Zheng, Ping; Sun, Jibin; Ma, Yanhe

    2017-11-16

    Corynebacterium glutamicum is an important industrial workhorse and advanced genetic engineering tools are urgently demanded. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) have revolutionized the field of genome engineering. The CRISPR/Cas9 system that utilizes NGG as protospacer adjacent motif (PAM) and has good targeting specificity can be developed into a powerful tool for efficient and precise genome editing of C. glutamicum. Herein, we developed a versatile CRISPR/Cas9 genome editing toolbox for C. glutamicum. Cas9 and gRNA expression cassettes were reconstituted to combat Cas9 toxicity and facilitate effective termination of gRNA transcription. Co-transformation of Cas9 and gRNA expression plasmids was exploited to overcome high-frequency mutation of cas9, allowing not only highly efficient gene deletion and insertion with plasmid-borne editing templates (efficiencies up to 60.0 and 62.5%, respectively) but also simple and time-saving operation. Furthermore, CRISPR/Cas9-mediated ssDNA recombineering was developed to precisely introduce small modifications and single-nucleotide changes into the genome of C. glutamicum with efficiencies over 80.0%. Notably, double-locus editing was also achieved in C. glutamicum. This toolbox works well in several C. glutamicum strains including the widely-used strains ATCC 13032 and ATCC 13869. In this study, we developed a CRISPR/Cas9 toolbox that could facilitate markerless gene deletion, gene insertion, precise base editing, and double-locus editing in C. glutamicum. The CRISPR/Cas9 toolbox holds promise for accelerating the engineering of C. glutamicum and advancing its application in the production of biochemicals and biofuels.

  17. La ponction biopsie hépatique à Dakar: indications, complications et apport diagnostique - à propos de 70 cas

    Science.gov (United States)

    Touré, Papa Souleymane; Léye, Abdoulaye; Diop, Madoky Maguette; Gueye, Mame Daouda; Léye, Yakham Mohamed; Berthé, Adama; Mourtalla Ka, Mamadou

    2014-01-01

    Introduction Les objectifs de notre travail étaient de déterminer les indications, les complications et l'apport diagnostique de la ponction biopsie hépatique (PBH) transpariétale. Méthodes Il s'agissait d'une étude rétrospective descriptive allant du janvier 2006 à décembre 2010, réalisée dans le service de Médecine Interne de l'hôpital de Pikine de Dakar. Etaient inclus, tous les malades ayant subi une biopsie hépatique, en ambulatoire ou en hospitalisation. Etaient exclus, tous les patients qui ont bénéficié d'une PBH dont les résultats n'ont pas été retrouvés. Les données suivantes étaient étudié: l’âge, le sexe, les indications, la taille du fragment biopsique, Le nombre de carottes, les complications, la comparaison des diagnostics pré biopsiques avec les comptes rendus histologiques. Résultats Ont été colligées 70 patients atteints d'hépatopathie chronique ayant bénéficiés d'une PBH. Il s'agissait de 46 hommes (65.71%) et 24 femmes (34.29%). L’âge moyen des patients était de 36 ans. Les PBH étaient réalisées en ambulatoire chez 58 patients (82.86%) et chez 12 malades hospitalisés (17,14%). Les indications étaient dominées par les hépatites virales chroniques dans 62,86% (44cas), suivi des processus tumoraux du foie dans 24.29% (17 cas). Les complications survenues chez 15 patients (21.43%) étaient représentées de 14 cas de douleur (20%) et d'un cas de malaise vagal (1.43%). Les 70 PBH effectuées ont ramené une carotte dans 35,71% des cas, 2 à 6 carottes dans 32,87% des cas. La longueur moyenne des fragments biopsiques était de 22 ±8 mm. Soixante-six résultats étaient interprétables et 4 non interprétables soit une performance diagnostique de 94,29%. Conclusion La PBH est de pratique sure, avec un respect des contres indications et une bonne maitrise de la technique. Son acceptabilité a été bonne dans notre pratique et sa rentabilité diagnostique excellente. Elle devrait être beaucoup plus

  18. Profil épidémiologique des hémoglobinopathies: étude transversale descriptive autour du cas index

    Science.gov (United States)

    Dahmani, Fatima; Benkirane, Souad; Kouzih, Jaafar; Woumki, Aziz; Mamad, Hassan; Masrar, Azlarab

    2017-01-01

    Les hémoglobinopathies sont des affections constitutionnelles conséquentes à des anomalies des hémoglobines. Elles sont souvent graves dans leurs formes majeures, leur prise en charge est lourde avec un grand impact psycho-social sur les patients et leur famille. Classées parmi les maladies rares, elles sont encore insuffisamment connues des professionnels de santé. Cette méconnaissance est à l'origine d'une errance diagnostique, d'un retard dans leur prise en charge et par conséquent une morbidité et une mortalité élevée chez ces patients. L'Organisation Mondiale de la Santé (OMS) a publié en 2008 des données concernant l'épidémiologie des hémoglobinopathies: plus de 330000 cas naissent chaque année avec une hémoglobinopathie (83% des cas de drépanocytose, 17% des cas de thalassémie). Les troubles de l'hémoglobine sont responsables d'environ 3,4% des décès chez les moins de 5 ans. A l'échelle mondiale, 7% environ des femmes enceintes seraient porteuses d'une forme de la thalassémie et 1% des couples sont à risque. Toutefois, elles sont relativement fréquentes dans certaines régions du globe où les mariages consanguins sont communs. Afin de décrire les caractéristiques épidémiologiques des familles à risque d'hémoglobinopathies (étude autour du cas) dont les cas index sont suivis au service de pédiatrie à l'Hôpital Provincial El Idrisi de Kenitra au Maroc, une étude transversale descriptive a été réalisée durant deux enquêtes la première en mai 2015 et la deuxième en juin de la même année lors des journées de vaccination des cas index contre le pneumocoque. Après avoir recueilli les données épidémiologiques de nos patients, nous avons réalisé une étude biologique comportant: l'hémogramme avec étude morphologique des globules rouges en coloration MGG et numération automatique des réticulocytes; les électrophorèses de l'hémoglobine à pH alcalin (8.8) et secondairement à pH acide (5.4) sur gel d

  19. Coupling Modified Linear Spectral Mixture Analysis and Soil Conservation Service Curve Number (SCS-CN Models to Simulate Surface Runoff: Application to the Main Urban Area of Guangzhou, China

    Directory of Open Access Journals (Sweden)

    Jianhui Xu

    2016-11-01

    Full Text Available Land surface characteristics, including soil type, terrain slope, and antecedent soil moisture, have significant impacts on surface runoff during heavy precipitation in highly urbanized areas. In this study, a Linear Spectral Mixture Analysis (LSMA method is modified to extract high-precision impervious surface, vegetation, and soil fractions. In the modified LSMA method, the representative endmembers are first selected by combining a high-resolution image from Google Earth; the unmixing results of the LSMA are then post-processed to reduce errors of misclassification with Normalized Difference Built-up Index (NDBI and Normalized Difference Vegetation Index (NDVI. The modified LSMA is applied to the Landsat 8 Operational Land Imager (OLI image from 18 October 2015 of the main urban area of Guangzhou city. The experimental result indicates that the modified LSMA shows improved extraction performance compared with the conventional LSMA, as it can significantly reduce the bias and root-mean-square error (RMSE. The improved impervious surface, vegetation, and soil fractions are used to calculate the composite curve number (CN for each pixel according to the Soil Conservation Service curve number (SCS-CN model. The composite CN is then adjusted with regional data of the terrain slope and total 5-day antecedent precipitation. Finally, the surface runoff is simulated with the SCS-CN model by combining the adjusted CN and real precipitation data at 1 p.m., 4 May 2015.

  20. Erysipèle du membre inférieur: étude de 400 cas [Erysipelas of the lower limb: study of 400 cases

    Directory of Open Access Journals (Sweden)

    Amina Aounallah

    2017-11-01

    Full Text Available Introduction: Erysipelas is an acute, non-necrotizing dermo-hypodermitis of predominantly streptococcal origin. Objective: To clarify the epidemiological and evolutionary features of the lower limb erysipelas through a hospital series. Materiels and Methods: We retrospectively analyzed all cases of lower limb erysipelas hospitalized in the Department of Dermatology of the Farhat Hached Hospital of Sousse between January 2000 and December 2015 (10 years. Results: Four hundred cases of erysipelas of the lower limb were recorded. The mean age of the patients was 55.82 years. The sex ratio was 1.51. The main predisposing factors were sedentarity, diabetes and obesity. In 96.75% of cases, entry lesion, like mycoses or traumatic injuries was noted. Clinically, a classic presentation of erysipelas was described in all cases. Erysipela was unilateral in 96% of cases. Treatment was based on intravenous penicillin G in 86.5% of cases, on an average of 9.75 days. The evolution was favorable in 83.25% of cases. Antibioprophylaxis was prescribed in 38% of cases. Loco-regional and general complications were noted in 10.25% of cases. Three patients died. Late complications were dominated by relapses and persistence of sequelled lymphedema. RÉSUMÉ Introduction: L’érysipèle est une dermo-hypodermite aigue, non nécrosante, d’origine principalement streptococcique. Objectif: préciser les particularités épidémio-cliniques et évolutives de l’Erysipèle du membre inferieur à travers une série hospitalière. Matériel et Méthodes: Nous avons rétrospectivement analysé tous les cas d’érysipèle du membre inférieur hospitalisés dans le service de dermatologie de l’hôpital Farhat Hached de Sousse entre janvier 2000 et décembre 2015 (10 ans. Résultats: Quatre cents cas d’érysipèle du membre inférieur ont été recensés. L’âge moyen des patients était de 55.82 ans. Le sexe ratio était de 1.51. Les principaux facteurs favorisants

  1. Le cancer différencié de la thyroïde chez l’enfant et l’adolescent: à propos de 22 cas

    Science.gov (United States)

    Anajar, Said; Tatari, Mohammed; Lakhbal, Adil; Abada, Reda; Rouadi, Sami; Roubal, Mohammed; Mahtar, Mohammed

    2017-01-01

    L’obectif était de mettre en relief les particularités du cancer de la thyroïde chez l’enfant et l’adolescent, et d’évaluer nos résultats par rapport à la littérature internationale a travers une série de cas la plus représentatif au Maroc: 22 cas. C'est une étude rétrospective descriptive des patients atteints de cancer différencié de la thyroïde, hospitalisés au service d’ORL et de Chirurgie Cervico-faciale de L’hopital 20 Août de Casablanca-Maroc, sur la période qui s’étend de Janvier 1995 à Mars 2015. Nous avons recueilli les données relatives à 22 cas, qui répondaient à nos critères d’inclusion. L’âge moyen de nos patients était de 14 ans, avec une sex-ratio 3,4, la plupart de nos patients ont consulté pour un nodule thyroïdien, associé dans 22,7% des cas à une adénopathie cervicale, et dans 9,1% à des signes de compression. L’ensemble des patients ont bénéficié d’une thyroïdectomie totale, suivie d’un curage ganglionnaire dans 31,82%. Le diagnostic de cancer thyroïdien a reposé sur l’examen anatomopathologique de la pièce opératoire, qui a objectivé un carcinome papillaire dans 95,4% des cas, et un carcinome vésiculaire dans 4,5%. Le traitement par l’iode radioactif 131 a été réalisé dans 100% des cas. Par la suite tous nos patients ont été mis sous hormonothérapie thyroïdienne. Une surveillance étroite et régulière a permis de détecter des métastases ganglionnaires chez 3 patients, et les métastases à distance chez 4 patients. Le cancer différencié de la thyroïde de l’enfant et l’adolescent est une entité rare mais agressive, son traitement se base sur la chirurgie, associée à l’irathérapie donnant un pronostic excellent. PMID:29255541

  2. Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors.

    Science.gov (United States)

    Hu, Jiabiao; Lei, Yong; Wong, Wing-Ki; Liu, Senquan; Lee, Kai-Chuen; He, Xiangjun; You, Wenxing; Zhou, Rui; Guo, Jun-Tao; Chen, Xiongfong; Peng, Xianlu; Sun, Hao; Huang, He; Zhao, Hui; Feng, Bo

    2014-04-01

    The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around -120 to -80 bp, while highly effective sgRNAs targeted from -147 to -89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells.

  3. DNA-Sequence Based Typing of the Cronobacter Genus Using MLST, CRISPR-cas Array and Capsular Profiling

    Directory of Open Access Journals (Sweden)

    Pauline Ogrodzki

    2017-09-01

    Full Text Available The Cronobacter genus is composed of seven species, within which a number of pathovars have been described. The most notable infections by Cronobacter spp. are of infants through the consumption of contaminated infant formula. The description of the genus has greatly improved in recent years through DNA sequencing techniques, and this has led to a robust means of identification. However some species are highly clonal and this limits the ability to discriminate between unrelated strains by some methods of genotyping. This article updates the application of three genotyping methods across the Cronobacter genus. The three genotyping methods were multilocus sequence typing (MLST, capsular profiling of the K-antigen and colanic acid (CA biosynthesis regions, and CRISPR-cas array profiling. A total of 1654 MLST profiled and 286 whole genome sequenced strains, available by open access at the PubMLST Cronobacter database, were used this analysis. The predominance of C. sakazakii and C. malonaticus in clinical infections was confirmed. The majority of clinical strains being in the C. sakazakii clonal complexes (CC 1 and 4, sequence types (ST 8 and 12 and C. malonaticus ST7. The capsular profile K2:CA2, previously proposed as being strongly associated with C. sakazakii and C. malonaticus isolates from severe neonatal infections, was also found in C. turicensis, C. dublinensis and C. universalis. The majority of CRISPR-cas types across the genus was the I-E (Ecoli type. Some strains of C. dublinensis and C. muytjensii encoded the I-F (Ypseudo type, and others lacked the cas gene loci. The significance of the expanding profiling will be of benefit to researchers as well as governmental and industrial risk assessors.

  4. DNA-Sequence Based Typing of the Cronobacter Genus Using MLST, CRISPR-cas Array and Capsular Profiling.

    Science.gov (United States)

    Ogrodzki, Pauline; Forsythe, Stephen J

    2017-01-01

    The Cronobacter genus is composed of seven species, within which a number of pathovars have been described. The most notable infections by Cronobacter spp. are of infants through the consumption of contaminated infant formula. The description of the genus has greatly improved in recent years through DNA sequencing techniques, and this has led to a robust means of identification. However some species are highly clonal and this limits the ability to discriminate between unrelated strains by some methods of genotyping. This article updates the application of three genotyping methods across the Cronobacter genus. The three genotyping methods were multilocus sequence typing (MLST), capsular profiling of the K -antigen and colanic acid (CA) biosynthesis regions, and CRISPR- cas array profiling. A total of 1654 MLST profiled and 286 whole genome sequenced strains, available by open access at the PubMLST Cronobacter database, were used this analysis. The predominance of C. sakazakii and C. malonaticus in clinical infections was confirmed. The majority of clinical strains being in the C. sakazakii clonal complexes (CC) 1 and 4, sequence types (ST) 8 and 12 and C. malonaticus ST7. The capsular profile K2:CA2, previously proposed as being strongly associated with C. sakazakii and C. malonaticus isolates from severe neonatal infections, was also found in C. turicensis , C. dublinensis and C. universalis . The majority of CRISPR- cas types across the genus was the I-E (Ecoli) type. Some strains of C. dublinensis and C. muytjensii encoded the I-F (Ypseudo) type, and others lacked the cas gene loci. The significance of the expanding profiling will be of benefit to researchers as well as governmental and industrial risk assessors.

  5. El cas estrany d'una nena amb tuberculosi renal

    OpenAIRE

    Martínez Roig, Antonio

    2009-01-01

    La tuberculosi és una afecció més pròpia de localitzar-se als pulmons, a través del contagi per via aèria, que no pas fora d'ells. La que es produeix als ronyons seria només un 3% i la infecció estaria vinculada a la disseminació per via sanguínia i limfàtica de la tuberculosi pulmonar. Si considerem que la tuberculosi es diagnostica principalment a l'adolescència, trobarem força peculiar el cas d'una nena que va desenvolupar una tuberculosi renal amb tretze mesos de vida. La nena patia una d...

  6. CAS CERN Accelerator School: Fourth general accelerator physics course

    International Nuclear Information System (INIS)

    Turner, S.

    1991-01-01

    The fourth CERN Accelerator School (CAS) basic course on General Accelerator Physics was given at KFA, Juelich, from 17 to 28 September 1990. Its syllabus was based on the previous similar courses held at Gif-sur-Yvette in 1984, Aarhus 1986, and Salamanca 1988, and whose proceedings were published as CERN Reports 85-19, 87-10, and 89-05, respectively. However, certain topics were treated in a different way, improved or extended, while new subjects were introduced. All of these appear in the present proceedings, which include lectures or seminars on the history and applications of accelerators, phase space and emittance, chromaticity, beam-beam effects, synchrotron radiation, radiation damping, tune measurement, transition, electron cooling, the designs of superconducting magnets, ring lattices, conventional RF cavities and ring RF systems, and an introduction to cyclotrons. (orig.)

  7. CAS Accelerators for Medical Applications in Vösendorf, Austria

    CERN Multimedia

    CERN Accelerator School

    2015-01-01

    The CERN Accelerator School (CAS) and MedAustron jointly organised a course on Accelerators for Medical Applications in Vösendorf, Austria between 26 May and 5 June 2015. The course was held at the Eventhotel Pyramide on the outskirts of Vienna, and was attended by 76 participants from 29 countries, coming from as far away as Canada, China, Lithuania, Thailand, Ukraine and Russia.       The intensive programme comprised 37 lectures. The emphasis was on using charged particle beams for cancer therapy and the programme began by covering the way in which particles interact with biological material, how this translates into the dose needed for treatment and how this dose is best delivered. The different accelerator options for providing the particles needed were then presented in some detail. The production of radioisotopes and how these are used for diagnostics and therapy was also covered, together with a look at novel acceleration techniques that may play a role i...

  8. CAS Accelerator Physics (High-Power Hadron Machines) in Spain

    CERN Multimedia

    CAS

    2011-01-01

    The CERN Accelerator School (CAS) and ESS-Bilbao jointly organised a specialised course on High-Power Hadron Machines, held at the Hotel Barceló Nervión in Bilbao, Spain, from 24 May to 2 June, 2011.   CERN Accelerator School students. After recapitulation lectures on the essentials of accelerator physics and review lectures on the different types of accelerators, the programme focussed on the challenges of designing and operating high-power facilities. The particular problems for RF systems, beam instrumentation, vacuum, cryogenics, collimators and beam dumps were examined. Activation of equipment, radioprotection and remote handling issues were also addressed. The school was very successful, with 69 participants of 22 nationalities. Feedback from the participants was extremely positive, praising the expertise and enthusiasm of the lecturers, as well as the high standard and excellent quality of their lectures. In addition to the academic programme, the participants w...

  9. CAS course on Advanced Accelerator Physics in Warsaw

    CERN Multimedia

    CERN Accelerator School

    2015-01-01

    The CERN Accelerator School (CAS) and the National Centre for Nuclear Research (NCBJ) recently organised a course on Advanced Accelerator Physics. The course was held in Warsaw, Poland from 27 September to 9 October 2015.    The course followed an established format with lectures in the mornings and practical courses in the afternoons. The lecture programme consisted of 34 lectures, supplemented by private study, tutorials and seminars. The practical courses provided ‘hands-on’ experience of three topics: ‘Beam Instrumentation and Diagnostics’, ‘RF Measurement Techniques’ and ‘Optics Design and Corrections’. Participants selected one of the three courses and followed their chosen topic throughout the duration of the school. Sixty-six students representing 18 nationalities attended this course, with most participants coming from European counties, but also from South Korea, Taiwan and Russia. Feedback from th...

  10. CAS CERN Accelerator School. Third advanced accelerator physics course

    International Nuclear Information System (INIS)

    Turner, S.

    1990-01-01

    The third version of the CERN Accelerator School's (CAS) advanced course on General Accelerator Physics was given at Uppsala University from 18-29 September, 1989. Its syllabus was based on the previous courses held in Oxford, 1985 and Berlin, 1987 whose proceedings were published as CERN Yellow Reports 87-03 and 89-01 respectively. However, the opportunity was taken to emphasize the physics of small accelerators and storage rings, to present some topics in new ways, and to introduce new seminars. Thus the lectures contained in the present volume include chromaticity, dynamic aperture, kinetic theory, Landau damping, ion-trapping, Schottky noise, laser cooling and small ring lattice problems while the seminars include interpretation of numerical tracking, internal targets and living with radiation. (orig.)

  11. CAS Accelerator Physics (RF for Accelerators) in Denmark

    CERN Multimedia

    Barbara Strasser

    2010-01-01

    The CERN Accelerator School (CAS) and Aarhus University jointly organised a specialised course on RF for Accelerators, at the Ebeltoft Strand Hotel, Denmark from 8 to 17 June 2010.   Caption The challenging programme focused on the introduction of the underlying theory, the study and the performance of the different components involved in RF systems, the RF gymnastics and RF measurements and diagnostics. This academic part was supplemented with three afternoons dedicated to practical hands-on exercises. The school was very successful, with 100 participants representing 25 nationalities. Feedback from the participants was extremely positive, praising the expertise and enthusiasm of the lecturers, as well as the high standard and excellent quality of their lectures. In addition to the academic programme, the participants were able to visit a small industrial exhibition organised by Aarhus University and take part in a one-day excursion consisting of a visit of the accelerators operated ...

  12. CAS course on advanced accelerator physics in Trondheim, Norway

    CERN Multimedia

    CERN Accelerator School

    2013-01-01

    The CERN Accelerator School (CAS) and the Norwegian University of Science and Technology (NTNU) recently organised a course on advanced accelerator physics. The course was held in Trondheim, Norway, from 18 to 29 August 2013. Accommodation and lectures were at the Hotel Britannia and practical courses were held at the university.   The course's format included lectures in the mornings and practical courses in the afternoons. The lecture programme consisted of 32 lectures supplemented by discussion sessions, private study and tutorials. The practical courses provided "hands-on" experience in three topics: RF measurement techniques, beam instrumentation and diagnostics, and optics design and corrections. Participants selected one of the three courses and followed the chosen topic throughout the course. The programme concluded with seminars and a poster session.  70 students representing 21 nationalities were selected from over 90 applicants, with most participa...

  13. Study of electronic and structural properties of CaS

    International Nuclear Information System (INIS)

    Mirfenderski, M.; Akbarzdeh, H.; Mokhtari, A.

    2003-01-01

    The electronic and structural properties of CaS are calculated using full potential linearized augmented plane wave method within the local density approximation and generalized gradient approximation for the exchange -correlation energy. For both structures, NaCl structure (B1) and CsCl structure (B2), the obtained values for lattice parameters, bulk modulus and its pressure derivative and transition pressure are in reasonable agreement with the experimental values. For electronic properties, the obtained value for band gap is smaller than the experimental value as well as other calculated results based on density functional theory. Engel and Vosko calculated an exchange potential for some atoms within the so-called optimize-potential model and then used the virial relation and constructed a new exchange-correlation functional. We used that functional and obtained reasonable results for band gap. Finally we investigated the possibility for a third phase ( Zinc Blend structure) for this crystal

  14. CAS course on Power Converters in Baden, Switzerland

    CERN Multimedia

    CERN Accelerator School

    2014-01-01

    The CERN Accelerator School (CAS) and the Paul Scherrer Institute (PSI) recently organised a specialised course on Power Converters, which was held at the Hotel du Parc in Baden, Switzerland from 7 to 14 May 2014.   Photo courtesy of Markus Fischer, Paul Scherrer Institut. Following some recapitulation lectures on accelerators and the requirements on power converters, the course covered a wide range of topics related to the different types of power converters needed for particle accelerators. Topical seminars completed the programme. The course was very successful, attended by 84 students representing 21 nationalities, mostly from European countries but also from America, Brazil, Canada, China, Iran, Jordan and Thailand. Feedback from the participants was very positive, reflecting the high standard of the lectures and teaching. In addition to the academic programme, the participants also had an opportunity to take part in a full-day site visit to ABB and PSI and an excursion to the Rhine Fall...

  15. CAS course on Intensity Limitations in Particle Beams at CERN

    CERN Multimedia

    CERN Accelerator School

    2015-01-01

    The CERN Accelerator School (CAS) recently organised a specialised course on Intensity Limitations in Particle Beams, at CERN from 2 to 11 November, 2015.     Many accelerators and storage rings, whether intended for particle physics experiments, synchrotron light sources or industrial applications, require beams of high brightness and the highest possible intensities. A good understanding of the possible limitations is required to achieve the desired performance. This course covered the interaction of beams with their surroundings and with other beams, as well as further collective effects. The lectures on the effects and possible mitigations were complemented by tutorials. The course was very successful, with 66 students representing 14 nationalities attending. Most participants came from European counties, but also from Armenia, China and Russia. Feedback from the participants was positive, reflecting the standard of the lectures and teaching. In addition to the academic pro...

  16. CAS CERN Accelerator School: Cyclotrons, linacs and their applications. Proceedings

    International Nuclear Information System (INIS)

    Turner, S.

    1996-01-01

    These proceedings present the lectures given at the eighth specialized course organized by the CERN Accelerator School (CAS), the topic this time being 'Cyclotrons, Linacs and Their Applications'. Following an introductory lecture on linacs, the fundamental features of electron, ion and induction linacs are described together with their RF systems and particle sources. Cyclotrons are then introduced followed by details of their different types, their magnet and RF design, and their injection and extraction systems, with a glance towards exotic and possible future machines. Chapters are then presented on the use of linacs and cyclotrons for medical, fission, fusion and material applications, as well as for isotope production. Finally, descriptions of the design of a radioisotope facility, the matching of accelerators to their task and the computational tools used in their design are included. (orig.)

  17. CAS CERN Accelerator School: Cyclotrons, linacs and their applications. Proceedings

    Energy Technology Data Exchange (ETDEWEB)

    Turner, S [ed.

    1996-03-04

    These proceedings present the lectures given at the eighth specialized course organized by the CERN Accelerator School (CAS), the topic this time being `Cyclotrons, Linacs and Their Applications`. Following an introductory lecture on linacs, the fundamental features of electron, ion and induction linacs are described together with their RF systems and particle sources. Cyclotrons are then introduced followed by details of their different types, their magnet and RF design, and their injection and extraction systems, with a glance towards exotic and possible future machines. Chapters are then presented on the use of linacs and cyclotrons for medical, fission, fusion and material applications, as well as for isotope production. Finally, descriptions of the design of a radioisotope facility, the matching of accelerators to their task and the computational tools used in their design are included. (orig.).

  18. 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.

    2014-03-01

    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.

  19. Unexpected heterogeneity derived from Cas9 ribonucleoprotein-introduced clonal cells at the HPRT1 locus.

    Science.gov (United States)

    Sakuma, Tetsushi; Mochida, Keiji; Nakade, Shota; Ezure, Toru; Minagawa, Sachi; Yamamoto, Takashi

    2018-04-01

    Single-cell cloning is an essential technique for establishing genome-edited cell clones mediated by programmable nucleases such as CRISPR-Cas9. However, residual genome-editing activity after single-cell cloning may cause heterogeneity in the clonal cells. Previous studies showed efficient mutagenesis and rapid degradation of CRISPR-Cas9 components in cultured cells by introducing Cas9 ribonucleoproteins (RNPs). In this study, we investigated how the timing for single-cell cloning of Cas9 RNP-transfected cells affected the heterogeneity of the resultant clones. We carried out transfection of Cas9 RNPs targeting several loci in the HPRT1 gene in HCT116 cells, followed by single-cell cloning at 24, 48, 72 hr and 1 week post-transfection. After approximately 3 weeks of incubation, the clonal cells were collected and genotyped by high-resolution microchip electrophoresis and Sanger sequencing. Unexpectedly, long-term incubation before single-cell cloning resulted in highly heterogeneous clones. We used a lipofection method for transfection, and the media containing transfectable RNPs were not removed before single-cell cloning. Therefore, the active Cas9 RNPs were considered to be continuously incorporated into cells during the precloning incubation. Our findings provide a warning that lipofection of Cas9 RNPs may cause continuous introduction of gene mutations depending on the experimental procedures. © 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

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

    Science.gov (United States)

    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.

  1. Spermatogenic Cell-Specific Gene Mutation in Mice via CRISPR-Cas9.

    Science.gov (United States)

    Bai, Meizhu; Liang, Dan; Wang, Yinghua; Li, Qing; Wu, Yuxuan; Li, Jinsong

    2016-05-20

    Tissue-specific knockout technology enables the analysis of the gene function in specific tissues in adult mammals. However, conventional strategy for producing tissue-specific knockout mice is a time- and labor-consuming process, restricting rapid study of the gene function in vivo. CRISPR-Cas9 system from bacteria is a simple and efficient gene-editing technique, which has enabled rapid generation of gene knockout lines in mouse by direct injection of CRISPR-Cas9 into zygotes. Here, we demonstrate CRISPR-Cas9-mediated spermatogenic cell-specific disruption of Scp3 gene in testes in one step. We first generated transgenic mice by pronuclear injection of a plasmid containing Hspa2 promoter driving Cas9 expression and showed Cas9 specific expression in spermatogenic cells. We then produced transgenic mice carrying Hspa2 promoter driven Cas9 and constitutive expressed sgRNA targeting Scp3 gene. Male founders were infertile due to developmental arrest of spermatogenic cells while female founders could produce progeny normally. Consistently, male progeny from female founders were infertile and females could transmit the transgenes to the next generation. Our study establishes a CRISPR-Cas9-based one-step strategy to analyze the gene function in adult tissues by a temporal-spatial pattern. Copyright © 2016 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    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.

  3. CRISPR/Cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus UTEX 2973.

    Science.gov (United States)

    Wendt, Kristen E; Ungerer, Justin; Cobb, Ryan E; Zhao, Huimin; Pakrasi, Himadri B

    2016-06-23

    As autotrophic prokaryotes, cyanobacteria are ideal chassis organisms for sustainable production of various useful compounds. The newly characterized cyanobacterium Synechococcus elongatus UTEX 2973 is a promising candidate for serving as a microbial cell factory because of its unusually rapid growth rate. Here, we seek to develop a genetic toolkit that enables extensive genomic engineering of Synechococcus 2973 by implementing a CRISPR/Cas9 editing system. We targeted the nblA gene because of its important role in biological response to nitrogen deprivation conditions. First, we determined that the Streptococcus pyogenes Cas9 enzyme is toxic in cyanobacteria, and conjugational transfer of stable, replicating constructs containing the cas9 gene resulted in lethality. However, after switching to a vector that permitted transient expression of the cas9 gene, we achieved markerless editing in 100 % of cyanobacterial exconjugants after the first patch. Moreover, we could readily cure the organisms of antibiotic resistance, resulting in a markerless deletion strain. High expression levels of the Cas9 protein in Synechococcus 2973 appear to be toxic and result in cell death. However, introduction of a CRISPR/Cas9 genome editing system on a plasmid backbone that leads to transient cas9 expression allowed for efficient markerless genome editing in a wild type genetic background.

  4. CRISPR/Cas9: the Jedi against the dark empire of diseases.

    Science.gov (United States)

    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.

  5. CRISPR-Cas9-Mediated Genome Editing and Transcriptional Control in Yarrowia lipolytica.

    Science.gov (United States)

    Schwartz, Cory; Wheeldon, Ian

    2018-01-01

    The discovery and adaptation of RNA-guided nucleases has resulted in the rapid development of efficient, scalable, and easily accessible synthetic biology tools for targeted genome editing and transcriptional control. In these systems, for example CRISPR-Cas9 from Streptococcus pyogenes, a protein with nuclease activity is targeted to a specific nucleotide sequence by a short RNA molecule, whereupon binding it cleaves the targeted nucleotide strand. To extend this genome-editing ability to the industrially important oleaginous yeast Yarrowia lipolytica, we developed a set of easily usable and effective CRISPR-Cas9 episomal vectors. In this protocols chapter, we first present a method by which arbitrary protein-coding genes can be disrupted via indel formation after CRISPR-Cas9 targeting. A second method demonstrates how the same CRISPR-Cas9 system can be used to induce markerless gene cassette integration into the genome by inducing homologous recombination after DNA cleavage by Cas9. Finally, we describe how a catalytically inactive form of Cas9 fused to a transcriptional repressor can be used to control transcription of native genes in Y. lipolytica. The CRISPR-Cas9 tools and strategies described here greatly increase the types of genome editing and transcriptional control that can be achieved in Y. lipolytica, and promise to facilitate more advanced engineering of this important oleaginous host.

  6. Occurrence and activity of a type II CRISPR-Cas system in Lactobacillus gasseri.

    Science.gov (United States)

    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.

  7. Supplementary Material for: CRISPR/Cas9-mediated viral interference in plants

    KAUST Repository

    Ali, Zahir; Abulfaraj, Aala A.; Idris, Ali; Ali, Shakila; Tashkandi, Manal; Mahfouz, Magdy

    2015-01-01

    Abstract Background The CRISPR/Cas9 system provides bacteria and archaea with molecular immunity against invading phages and conjugative plasmids. Recently, CRISPR/Cas9 has been used for targeted genome editing in diverse eukaryotic species. Results In this study, we investigate whether the CRISPR/Cas9 system could be used in plants to confer molecular immunity against DNA viruses. We deliver sgRNAs specific for coding and non-coding sequences of tomato yellow leaf curl virus (TYLCV) into Nicotiana benthamiana plants stably overexpressing the Cas9 endonuclease, and subsequently challenge these plants with TYLCV. Our data demonstrate that the CRISPR/Cas9 system targeted TYLCV for degradation and introduced mutations at the target sequences. All tested sgRNAs exhibit interference activity, but those targeting the stem-loop sequence within the TYLCV origin of replication in the intergenic region (IR) are the most effective. N. benthamiana plants expressing CRISPR/Cas9 exhibit delayed or reduced accumulation of viral DNA, abolishing or significantly attenuating symptoms of infection. Moreover, this system could simultaneously target multiple DNA viruses. Conclusions These data establish the efficacy of the CRISPR/Cas9 system for viral interference in plants, thereby extending the utility of this technology and opening the possibility of producing plants resistant to multiple viral infections.

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

    Science.gov (United States)

    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.

  9. 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.

  10. Primary processing of CRISPR RNA by the endonuclease Cas6 in Staphylococcus epidermidis.

    Science.gov (United States)

    Wakefield, Noelle; Rajan, Rakhi; Sontheimer, Erik J

    2015-10-07

    In many bacteria and archaea, an adaptive immune system (CRISPR-Cas) provides immunity against foreign genetic elements. This system uses CRISPR RNAs (crRNAs) derived from the CRISPR array, along with CRISPR-associated (Cas) proteins, to target foreign nucleic acids. In most CRISPR systems, endonucleolytic processing of crRNA precursors (pre-crRNAs) is essential for the pathway. Here we study the Cas6 endonuclease responsible for crRNA processing in the Type III-A CRISPR-Cas system from Staphylococcus epidermidis RP62a, a model for Type III-A CRISPR-Cas systems, and define substrate requirements for SeCas6 activity. We find that SeCas6 is necessary and sufficient for full-length crRNA biogenesis in vitro, and that it relies on both sequence and stem-loop structure in the 3' half of the CRISPR repeat for recognition and processing. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  11. 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...

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

    Directory of Open Access Journals (Sweden)

    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.

  13. CRISPRscan: designing highly efficient sgRNAs for CRISPR/Cas9 targeting in vivo

    Science.gov (United States)

    Moreno-Mateos, Miguel A.; Vejnar, Charles E.; Beaudoin, Jean-Denis; Fernandez, Juan P.; Mis, Emily K.; Khokha, Mustafa K.; Giraldez, Antonio J.

    2015-01-01

    CRISPR/Cas9 technology provides a powerful system for genome engineering. However, variable activity across different single guide RNAs (sgRNAs) remains a significant limitation. We have analyzed the molecular features that influence sgRNA stability, activity and loading into Cas9 in vivo. We observe that guanine enrichment and adenine depletion increase sgRNA stability and activity, while loading, nucleosome positioning and Cas9 off-target binding are not major determinants. We additionally identified truncated and 5′ mismatch-containing sgRNAs as efficient alternatives to canonical sgRNAs. Based on these results, we created a predictive sgRNA-scoring algorithm (CRISPRscan.org) that effectively captures the sequence features affecting Cas9/sgRNA activity in vivo. Finally, we show that targeting Cas9 to the germ line using a Cas9-nanos-3′-UTR fusion can generate maternal-zygotic mutants, increase viability and reduce somatic mutations. Together, these results provide novel insights into the determinants that influence Cas9 activity and a framework to identify highly efficient sgRNAs for genome targeting in vivo. PMID:26322839

  14. Interstellar and ejecta dust in the cas a supernova remnant

    Energy Technology Data Exchange (ETDEWEB)

    Arendt, Richard G. [CRESST, University of Maryland, Baltimore County, Baltimore, MD 21250 (United States); Dwek, Eli; Kober, Gladys [NASA Goddard Space Flight Center, Code 665, Greenbelt, MD 20771 (United States); Rho, Jeonghee [SETI Institute, 189 Bernardo Avenue, Mountain View, CA 94043 (United States); Hwang, Una, E-mail: Richard.G.Arendt@nasa.gov [NASA Goddard Space Flight Center, Code 662, Greenbelt, MD 20771 (United States)

    2014-05-01

    Infrared continuum observations provide a means of investigating the physical composition of the dust in the ejecta and swept up medium of the Cas A supernova remnant (SNR). Using low-resolution Spitzer IRS spectra (5-35 μm), and broad-band Herschel PACS imaging (70, 100, and 160 μm), we identify characteristic dust spectra, associated with ejecta layers that underwent distinct nuclear burning histories. The most luminous spectrum exhibits strong emission features at ∼9 and 21 μm and is closely associated with ejecta knots with strong Ar emission lines. The dust features can be reproduced by magnesium silicate grains with relatively low Mg to Si ratios. Another dust spectrum is associated with ejecta having strong Ne emission lines. It has no indication of any silicate features and is best fit by Al{sub 2}O{sub 3} dust. A third characteristic dust spectrum shows features that are best matched by magnesium silicates with a relatively high Mg to Si ratio. This dust is primarily associated with the X-ray-emitting shocked ejecta, but it is also evident in regions where shocked interstellar or circumstellar material is expected. However, the identification of dust composition is not unique, and each spectrum includes an additional featureless dust component of unknown composition. Colder dust of indeterminate composition is associated with emission from the interior of the SNR, where the reverse shock has not yet swept up and heated the ejecta. Most of the dust mass in Cas A is associated with this unidentified cold component, which is ≲ 0.1 M {sub ☉}. The mass of warmer dust is only ∼0.04 M {sub ☉}.

  15. The european emergency number 112 - the questionnaire

    Directory of Open Access Journals (Sweden)

    Krzysztof Goniewicz

    2017-07-01

    Conclusions. Most of the respondents (92% identify the 112 number as an emergency number that allows them to connect to emergency services from anywhere in the European Union. A significant number of respondents (47% identify the 112 number as an emergency number in Poland. One in three respondents will use the 999 number to contact the emergency services as a witness to an emergency in Poland. Non-medical university students more often (63% will use the 112 emergency number than medical college students (41%. Respondents (98% confirmed the usefulness of a unified emergency number throughout Europe, but decided that they were not sufficiently informed about 112 as the European emergency number.

  16. Peningkatan Nilai-Nilai Karakter Dengan Metode Mendongeng CAS CIS CUS Di BA Aisyiyah Kaponan 2 Ponorogo

    Directory of Open Access Journals (Sweden)

    Sidik Nuryanto

    2017-03-01

    Full Text Available Tujuan penelitian ini adalah melihat pelaksanaan mendongeng CAS CIS CUS dan peningkatan nilai karakter di BA ‘Aisyiyah Kaponan 2 Ponorogo. Penelitian ini dilaksanakan dengan subjek sejumlah 18 peserta didik. Metode penelitian menggunakan penelitian tindakan kelas. Prosedurnya meliputi perencanaan, pelaksanaan, pengamatan dan refleksi. Sumber data berupa informan, tempat dan peristiwa, dokumen dan arsip. Metode pengumpulan data dengan teknik wawancara, observasi, dan dokumentasi. Uji validitas data yang digunakan dengan teknik triangulasi. Data dianalisis dengan menggunakan model deskriptif komparatif.       Hasilnya pada kegiatan mendongeng dibagi menjadi 3 tahapan yaitu CAS CIS CUS. CAS (Cipta Aksi Super sebagai sarana untuk membuka dongeng, CIS (Cipta Inspirasi Super sebagai inti dari pelaksanaan dongeng, dan CUS (Cipta Usulan Super sebagai penutup dongeng. Pada pra siklus nilai karakter kecintaan terhadap Tuhan yang mulanya 68,8% pada siklus I menjadi 75%, dan pada siklus II 87,5%. Nilai disiplin anak pada kondisi awal 62,5% meningkat menjadi 68,8% dan akhir siklus II mencapai 93,8%. Nilai kemandirian terus meningkat dari 68,8% lalu 81,3% dan menjadi 93,8%. Nilai kerjasama bermula dari 56,3% pada siklus I menjadi 75% dan siklus II menjadi 81,3%. Tanggungjawab pada keadaan awal 56,3% berubah menjadi 68,8% serta siklus II menjadi 87,5%.The purpose of this study is to see the implementation of the CIS CAS CUS storytelling and character value increase in BA 'Aisyiyah Kaponan 2 Ponorogo. This study was conducted with 18 subjects number of learners. The research method uses classroom action research. The procedure includes the planning, implementation, observation and reflection. Sources of data in the form of informants, places and events, documents and archives. Data were collected by interview, observation and documentation. Test the validity of the data used by triangulation techniques. Data were analyzed using descriptive comparative

  17. Hypo-fractionated radiotherapy of breast cancer: long term results of a set of 80 cases treated in the radiotherapy department of the Oran university hospital; Radiotherapie hypofractionnee dans le cancer du sein: resultats a long terme d'une serie de 80 cas traites dans le service de radiotherapie du centre hospitalier universitaire d'Oran

    Energy Technology Data Exchange (ETDEWEB)

    Boukerche, A.; Yahia, A.; Madouri, R.; Belmiloud, H.; Dali-Youcef, A.F. [Service de radiotherapie, CHU d' Oran, Oran (Algeria)

    2011-10-15

    The authors report the assessment of the local and locoregional control and of the acute and late toxicity of adjuvant hypo-fractionated radiotherapy in breast cancer treatment. During 1998, 80 women have been treated by conservative or radical surgery and hypo-fractionated tele-cobalto-therapy (36 Gy in five fractions of 3 Gy a week, and a boost of 15 Gy in five fractions in case of conservative surgery). Results are discussed in terms of local and locoregional recurrence, tolerance, late toxicity, global survival, and tumour classification. The irradiation scheme seems perfectly achievable but a greater number of patients and a longer follow-up are required to better assess the efficiency and aesthetic results. Short communication

  18. Peptide/Cas9 nanostructures for ribonucleoprotein cell membrane transport and gene edition.

    Science.gov (United States)

    Lostalé-Seijo, Irene; Louzao, Iria; Juanes, Marisa; Montenegro, Javier

    2017-12-01

    The discovery of RNA guided endonucleases has emerged as one of the most important tools for gene edition and biotechnology. The selectivity and simplicity of the CRISPR/Cas9 strategy allows the straightforward targeting and editing of particular loci in the cell genome without the requirement of protein engineering. However, the transfection of plasmids encoding the Cas9 and the guide RNA could lead to undesired permanent recombination and immunogenic responses. Therefore, the direct delivery of transient Cas9 ribonucleoprotein constitutes an advantageous strategy for gene edition and other potential therapeutic applications of the CRISPR/Cas9 system. The covalent fusion of Cas9 with penetrating peptides requires multiple incubation steps with the target cells to achieve efficient levels of gene edition. These and other recent reports suggested that covalent conjugation of the anionic Cas9 ribonucleoprotein to cationic peptides would be associated with a hindered nuclease activity due to undesired electrostatic interactions. We here report a supramolecular strategy for the direct delivery of Cas9 by an amphiphilic penetrating peptide that was prepared by a hydrazone bond formation between a cationic peptide scaffold and a hydrophobic aldehyde tail. The peptide/protein non-covalent nanoparticles performed with similar efficiency and less toxicity than one of the best methods described to date. To the best of our knowledge this report constitutes the first supramolecular strategy for the direct delivery of Cas9 using a penetrating peptide vehicle. The results reported here confirmed that peptide amphiphilic vectors can deliver Cas9 in a single incubation step, with good efficiency and low toxicity. This work will encourage the search and development of conceptually new synthetic systems for transitory endonucleases direct delivery.

  19. Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress.

    Science.gov (United States)

    LeBlanc, Chantal; Zhang, Fei; Mendez, Josefina; Lozano, Yamile; Chatpar, Krishna; Irish, Vivian F; Jacob, Yannick

    2018-01-01

    The CRISPR/Cas9 system has greatly improved our ability to engineer targeted mutations in eukaryotic genomes. While CRISPR/Cas9 appears to work universally, the efficiency of targeted mutagenesis and the adverse generation of off-target mutations vary greatly between different organisms. In this study, we report that Arabidopsis plants subjected to heat stress at 37°C show much higher frequencies of CRISPR-induced mutations compared to plants grown continuously at the standard temperature (22°C). Using quantitative assays relying on green fluorescent protein (GFP) reporter genes, we found that targeted mutagenesis by CRISPR/Cas9 in Arabidopsis is increased by approximately 5-fold in somatic tissues and up to 100-fold in the germline upon heat treatment. This effect of temperature on the mutation rate is not limited to Arabidopsis, as we observed a similar increase in targeted mutations by CRISPR/Cas9 in Citrus plants exposed to heat stress at 37°C. In vitro assays demonstrate that Cas9 from Streptococcus pyogenes (SpCas9) is more active in creating double-stranded DNA breaks at 37°C than at 22°C, thus indicating a potential contributing mechanism for the in vivo effect of temperature on CRISPR/Cas9. This study reveals the importance of temperature in modulating SpCas9 activity in eukaryotes, and provides a simple method to increase on-target mutagenesis in plants using CRISPR/Cas9. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  20. Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation.

    Science.gov (United States)

    Mekler, Vladimir; Minakhin, Leonid; Severinov, Konstantin

    2017-05-23

    The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves double-stranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA-DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/guide RNA-DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs.

  1. The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET

    Directory of Open Access Journals (Sweden)

    Mengyi Yang

    2018-01-01

    Full Text Available Summary: Off-target binding and cleavage by Cas9 pose major challenges in its application. How the conformational dynamics of Cas9 govern its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single-molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms spontaneously transits among three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We also uncovered surprising long-range allosteric communication between the HNH domain and the RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated that a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the PAM-distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox. : Yang et al. revealed significant conformational dynamics of Cas9 at global and local scales using single-molecule FRET. They uncovered surprising long-range allosteric communication between the HNH nuclease domain and the RNA/DNA heteroduplex at the PAM-distal end that serves as a proofreading checkpoint to govern the nuclease activity and specificity of Cas9. Keywords: CRISPR, Cas9, single-molecule, FRET, conformational dynamics, proofreading, off-target, allosteric communication, genome editing

  2. Efficiently sampling conformations and pathways using the concurrent adaptive sampling (CAS) algorithm

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Surl-Hee; Grate, Jay W.; Darve, Eric F.

    2017-08-21

    Molecular dynamics (MD) simulations are useful in obtaining thermodynamic and kinetic properties of bio-molecules but are limited by the timescale barrier, i.e., we may be unable to efficiently obtain properties because we need to run microseconds or longer simulations using femtoseconds time steps. While there are several existing methods to overcome this timescale barrier and efficiently sample thermodynamic and/or kinetic properties, problems remain in regard to being able to sample un- known systems, deal with high-dimensional space of collective variables, and focus the computational effort on slow timescales. Hence, a new sampling method, called the “Concurrent Adaptive Sampling (CAS) algorithm,” has been developed to tackle these three issues and efficiently obtain conformations and pathways. The method is not constrained to use only one or two collective variables, unlike most reaction coordinate-dependent methods. Instead, it can use a large number of collective vari- ables and uses macrostates (a partition of the collective variable space) to enhance the sampling. The exploration is done by running a large number of short simula- tions, and a clustering technique is used to accelerate the sampling. In this paper, we introduce the new methodology and show results from two-dimensional models and bio-molecules, such as penta-alanine and triazine polymer

  3. Les tumeurs desmoides de la paroi thoracique : à propos de 12 cas Dermoid tumors of the thoracic wall: about 12 cases

    Directory of Open Access Journals (Sweden)

    Abdellatif Benosman

    2009-11-01

    Full Text Available Les tumeurs desmoides sont des tumeurs rares des tissus mous qui peuvent être très agressives localement. A travers l’expérience de notre service, nous analyserons les résultats du traitement chirurgical de ces tumeurs. De 1980 à 2008, 12 patients ont été opérés pour tumeur desmoide de la paroi thoracique. Le diagnostic a été suspecté sur la base des signes cliniques et radiologiques. Aucun patient n’avait un syndrome de Gardner. L’abord chirurgical a été souvent électif à l’aplomb de la tumeur. La résection a été complète dans 11 cas. La confirmation diagnostique a été apportée par l’étude histologique de la pièce opératoire. La durée du suivi post opératoire variait entre 24 et 180 mois. Une patiente était décédée par insuffisance cardiaque et rénale. 7 cas avaient récidivé, et qui ont été traités par simple résection complète dans 5 cas, dont un avait nécessité une greffe myocutanée ; par ailleurs, deux cas ont été traités par résection incomplète associée à une radiothérapie adjuvante. La chirurgie des tumeurs desmoides de la paroi thoracique doit être aussi large que possible pour diminuer le risque de récidive.

  4. Cell-type-specific genome editing with a microRNA-responsive CRISPR-Cas9 switch.

    Science.gov (United States)

    Hirosawa, Moe; Fujita, Yoshihiko; Parr, Callum J C; Hayashi, Karin; Kashida, Shunnichi; Hotta, Akitsu; Woltjen, Knut; Saito, Hirohide

    2017-07-27

    The CRISPR-Cas9 system is a powerful genome-editing tool useful in a variety of biotechnology and biomedical applications. Here we developed a synthetic RNA-based, microRNA (miRNA)-responsive CRISPR-Cas9 system (miR-Cas9 switch) in which the genome editing activity of Cas9 can be modulated through endogenous miRNA signatures in mammalian cells. We created miR-Cas9 switches by using a miRNA-complementary sequence in the 5΄-UTR of mRNA encoding Streptococcus pyogenes Cas9. The miR-21-Cas9 or miR-302-Cas9 switches selectively and efficiently responded to miR-21-5p in HeLa cells or miR-302a-5p in human induced pluripotent stem cells, and post-transcriptionally attenuated the Cas9 activity only in the target cells. Moreover, the miR-Cas9 switches could differentially control the genome editing by sensing endogenous miRNA activities within a heterogeneous cell population. Our miR-Cas9 switch system provides a promising framework for cell-type selective genome editing and cell engineering based on intracellular miRNA information. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. The Impact of DNA Topology and Guide Length on Target Selection by a Cytosine-Specific Cas9.

    Science.gov (United States)

    Tsui, Tsz Kin Martin; Hand, Travis H; Duboy, Emily C; Li, Hong

    2017-06-16

    Cas9 is an RNA-guided DNA cleavage enzyme being actively developed for genome editing and gene regulation. To be cleaved by Cas9, a double stranded DNA, or the protospacer, must be complementary to the guide region, typically 20-nucleotides in length, of the Cas9-bound guide RNA, and adjacent to a short Cas9-specific element called Protospacer Adjacent Motif (PAM). Understanding the correct juxtaposition of the protospacer- and PAM-interaction with Cas9 will enable development of versatile and safe Cas9-based technology. We report identification and biochemical characterization of Cas9 from Acidothermus cellulolyticus (AceCas9). AceCas9 depends on a 5'-NNNCC-3' PAM and is more efficient in cleaving negative supercoils than relaxed DNA. Kinetic as well as in vivo activity assays reveal that AceCas9 achieves optimal activity when combined with a guide RNA containing a 24-nucleotide complementarity region. The cytosine-specific, DNA topology-sensitive, and extended guide-dependent properties of AceCas9 may be explored for specific genome editing applications.

  6. Cell-type-specific genome editing with a microRNA-responsive CRISPR–Cas9 switch

    Science.gov (United States)

    Hirosawa, Moe; Fujita, Yoshihiko; Parr, Callum J. C.; Hayashi, Karin; Kashida, Shunnichi; Hotta, Akitsu; Woltjen, Knut

    2017-01-01

    Abstract The CRISPR–Cas9 system is a powerful genome-editing tool useful in a variety of biotechnology and biomedical applications. Here we developed a synthetic RNA-based, microRNA (miRNA)-responsive CRISPR–Cas9 system (miR-Cas9 switch) in which the genome editing activity of Cas9 can be modulated through endogenous miRNA signatures in mammalian cells. We created miR-Cas9 switches by using a miRNA-complementary sequence in the 5΄-UTR of mRNA encoding Streptococcus pyogenes Cas9. The miR-21-Cas9 or miR-302-Cas9 switches selectively and efficiently responded to miR-21-5p in HeLa cells or miR-302a-5p in human induced pluripotent stem cells, and post-transcriptionally attenuated the Cas9 activity only in the target cells. Moreover, the miR-Cas9 switches could differentially control the genome editing by sensing endogenous miRNA activities within a heterogeneous cell population. Our miR-Cas9 switch system provides a promising framework for cell-type selective genome editing and cell engineering based on intracellular miRNA information. PMID:28525578

  7. ON AN EXPONENTIAL INEQUALITY AND A STRONG LAW OF LARGE NUMBERS FOR MONOTONE MEASURES

    Czech Academy of Sciences Publication Activity Database

    Agahi, H.; Mesiar, Radko

    2014-01-01

    Roč. 50, č. 5 (2014), s. 804-813 ISSN 0023-5954 Institutional support: RVO:67985556 Keywords : Choquet expectation * a strong law of large numbers * exponential inequality * monotone probability Subject RIV: BA - General Mathematics Impact factor: 0.541, year: 2014 http://library.utia.cas.cz/separaty/2014/E/mesiar-0438052.pdf

  8. Inhibition of hepatitis B virus replication via HBV DNA cleavage by Cas9 from Staphylococcus aureus.

    Science.gov (United States)

    Liu, Yu; Zhao, Miaoxian; Gong, Mingxing; Xu, Ying; Xie, Cantao; Deng, Haohui; Li, Xueying; Wu, Hongkai; Wang, Zhanhui

    2018-04-01

    Chronic hepatitis B virus (HBV) infection is difficult to cure due to the presence of covalently closed circular DNA (cccDNA). Accumulating evidence indicates that the CRISPR/Cas9 system effectively disrupts HBV genome, including cccDNA, in vitro and in vivo. However, efficient delivery of CRISPR/Cas9 system to the liver or hepatocytes using an adeno-associated virus (AAV) vector remains challenging due to the large size of Cas9 from Streptococcus pyogenes (Sp). The recently identified Cas9 protein from Staphylococcus aureus (Sa) is smaller than SpCas9 and thus is able to be packaged into the AAV vector. To examine the efficacy of SaCas9 system on HBV genome destruction, we designed 5 guide RNAs (gRNAs) that targeted different HBV genotypes, 3 of which were shown to be effective. The SaCas9 system significantly reduced HBV antigen expression, as well as pgRNA and cccDNA levels, in Huh7, HepG2.2.15 and HepAD38 cells. The dual expression of gRNAs/SaCas9 in these cell lines resulted in more efficient HBV genome cleavage. In the mouse model, hydrodynamic injection of gRNA/SaCas9 plasmids resulted in significantly lower levels of HBV protein expression. We also delivered the SaCas9 system into mice with persistent HBV replication using an AAV vector. Both the AAV vector and the mRNA of Cas9 could be detected in the C3H mouse liver cells. Decreased hepatitis B surface antigen (HBsAg), HBV DNA and pgRNA levels were observed when a higher titer of AAV was injected, although this decrease was not significantly different from the control. In summary, the SaCas9 system accurately and efficiently targeted the HBV genome and inhibited HBV replication both in vitro and in vivo. The system was delivered by an AAV vector and maybe used as a novel therapeutic strategy against chronic HBV infection. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. Research Progress in the CAS Action Plan for the Development of Western China

    Institute of Scientific and Technical Information of China (English)

    Feng Renguo

    2005-01-01

    @@ To speed up the regional development in central and western China is a strategic decision made by the Chinese government at the turn of the century. For CAS research professionals, active participation into the campaign is a solemn historic commitment and a major task of the CAS-piloted national Knowledge Innovation Program. In early 2000, the CAS leadership formulated an Action Plan for Western China Development and initiated a research program aiming at the environmental evolution,ecological restoration and the sustainable exploitation of the local resources in the region.

  10. Efficient Oligo nucleotide mediated CRISPR-Cas9 Gene Editing in Aspergilli

    DEFF Research Database (Denmark)

    Nødvig, Christina Spuur; Hoof, Jakob Blæsbjerg; Kogle, Martin Engelhard

    2018-01-01

    CRISPR-Cas9 technologies are revolutionizing fungal gene editing. Here we show that survival of specific Cas9/sgRNA mediated DNA double strand breaks (DSBs) depends on the non-homologous end-joining, NHEJ, DNA repair pathway and we use this observation to develop a tool to assess protospacer....... niger, and in A. oryzae indicating that this type of repair may be wide spread in filamentous fungi. Importantly, we demonstrate that by using single-stranded oligo nucleotides for CRISPR-Cas9 mediated gene editing it is possible to introduce specific point mutations as well gene deletions...

  11. Dynamics of Indel Profiles Induced by Various CRISPR/Cas9 Delivery Methods

    DEFF Research Database (Denmark)

    Kosicki, Michael; Rajan, Sandeep S; Lorenzetti, Flaminia C

    2017-01-01

    The introduction of CRISPR/Cas9 gene editing in mammalian cells is a scientific breakthrough, which has greatly affected basic research and gene therapy. The simplicity and general access to CRISPR/Cas9 reagents has in an unprecedented manner "democratized" gene targeting in biomedical research...... approach. In this study we review the most commonly used indel detection methods and using a robust, sensitive, and cost efficient Indel Detection by Amplicon Analysis method, we have investigated the impact of the most commonly used CRISPR/Cas9 delivery formats, including lentivirus transduction, plasmid...

  12. Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Jakociunas, Tadas; Bonde, Ida; Herrgard, Markus

    2015-01-01

    CRISPR/Cas9 is a simple and efficient tool for targeted and marker-free genome engineering. Here, we report the development and successful application of a multiplex CRISPR/Cas9 system for genome engineering of up to 5 different genomic loci in one transformation step in baker's yeast Saccharomyces...... cerevisiae. To assess the specificity of the tool we employed genome re-sequencing to screen for off-target sites in all single knock-out strains targeted by different gRNAs. This extensive analysis identified no more genome variants in CRISPR/Cas9 engineered strains compared to wild-type reference strains...

  13. 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.

  14. Estudi de cas Unilever : la comunicació interna com a factor diferencial a les empreses

    OpenAIRE

    López Albrich, Alba; Universitat Autònoma de Barcelona. Facultat de Ciències de la Comunicació

    2014-01-01

    La comunicació interna i la gestió dels actius intangibles ha començat a prendre força recentment. Aquest estudi de cas és una investigació documental d’alguns dels factors que determinen una bona comunicació interna perquè aquesta esdevingui un avantatge per l’empresa. Amb aquests indicadors localitzats comprovar-ne la seva aplicació en un cas d'èxit de comunicació interna: el cas Unilever Espanya. I d'aquesta manera constatar el paper de la comunicació interna com a eina gestora estratègica...

  15. Epithelioma Spinocellulaire sur Cicatrice de Brulure (a Propos de Cinq Cas)

    Science.gov (United States)

    Tourabi, K.; Mejjati, H.; Ribqg, Y.; Achbouk, A.; Arrob, A.; Moussaoui, A.; Ihrai, H.

    2009-01-01

    Summary Pour étudier l'ulcère de Marjolin, tous les dossiers des patients qui se sont présentés pendant une période quinquennale à un Service de Chirurgie Plastique et des Brûlés au Maroc ayant un cancer sur cicatrice de brûlure, objectivé par un examen histopathologique, ont été inclus dans une fiche de recueil de données comprenant des paramètres liés à l'identité du malade, à l'inventaire préthérapeutique, au traitement et à l'évolution du cas. Les Auteurs, après avoir présenté les données des cinq patients inclus dans l'étude, considèrent les problèmes posés par les épithéliomas spinocellulaires sur cicatrice de brûlure, qui sont des affections graves et rares. La dégénérescence des cicatrices de brûlures est une évolution dramatique, causée par la négligence. Le traitement préventif par exérèse systématique de toute lésion suspecte doit être fortement souligné puisqu'il garantit la guérison quasi certaine. PMID:21991185

  16. Development and application of knowledge-based subject group integration platforms:A case study of Shanghai Institute of Ceramics,CAS

    Institute of Scientific and Technical Information of China (English)

    Yu; LIU; Jian; FU; Huijun; ZHENG; Hao; CHEN; Zhiping; YANG

    2014-01-01

    Purpose:According to the different requirements of research group users,we established the knowledge-based subject group integration platforms of Shanghai Institute of Ceramics,the Chinese Academy of Sciences(abbreviated as SIC CAS hereinafter),which were designed and constructed to better meet the needs of CAS research groups for their development,collaboration and communication.Design/methodology/approach:We first identified the requirements of users via preliminary investigation,and then chose CASI1 P,iLibrary and XKE technology,respectively as the building tools compatible with the major demands of users.These steps helped us complete the layout design of SIC CAS integration platforms,as well as its knowledge organization and integration.Findings:According to the need of users,we applied three types of platform construction technologies to five SIC integration platforms,and formulated standard norms for the further construction process,which could provide useful reference for a sustainable development for the extensive construction in CAS institutes.Research limitations:In order to make the SIC integration platforms more intelligent and have more functions,we need to enlarge the scale of the Platforms and upgrade the building tools for the platform construction.Practical implications:The nature of SIC sub-project integration platforms is to construct a content-sensitive environment which can embed knowledge services and knowledge applications seamlessly into scientific activities,so the Platform is expected to be a useful tool to help researchers better understand the recent development of the research field and form collaborations with their peers.Originality/value:SIC integration platforms are the only pilot construction that used 3different platform technologies in the first batch of knowledge-based subject group integration platforms of the Chinese Academy of Sciences.The construction is user-centered throughout the whole process,namely,from the technology

  17. On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires

    Directory of Open Access Journals (Sweden)

    Sukrit Silas

    2017-07-01

    Full Text Available Cas1 integrase is the key enzyme of the clustered regularly interspaced short palindromic repeat (CRISPR-Cas adaptation module that mediates acquisition of spacers derived from foreign DNA by CRISPR arrays. In diverse bacteria, the cas1 gene is fused (or adjacent to a gene encoding a reverse transcriptase (RT related to group II intron RTs. An RT-Cas1 fusion protein has been recently shown to enable acquisition of CRISPR spacers from RNA. Phylogenetic analysis of the CRISPR-associated RTs demonstrates monophyly of the RT-Cas1 fusion, and coevolution of the RT and Cas1 domains. Nearly all such RTs are present within type III CRISPR-Cas loci, but their phylogeny does not parallel the CRISPR-Cas type classification, indicating that RT-Cas1 is an autonomous functional module that is disseminated by horizontal gene transfer and can function with diverse type III systems. To compare the sequence pools sampled by RT-Cas1-associated and RT-lacking CRISPR-Cas systems, we obtained samples of a commercially grown cyanobacterium—Arthrospira platensis. Sequencing of the CRISPR arrays uncovered a highly diverse population of spacers. Spacer diversity was particularly striking for the RT-Cas1-containing type III-B system, where no saturation was evident even with millions of sequences analyzed. In contrast, analysis of the RT-lacking type III-D system yielded a highly diverse pool but reached a point where fewer novel spacers were recovered as sequencing depth was increased. Matches could be identified for a small fraction of the non-RT-Cas1-associated spacers, and for only a single RT-Cas1-associated spacer. Thus, the principal source(s of the spacers, particularly the hypervariable spacer repertoire of the RT-associated arrays, remains unknown.

  18. Les masques trompeurs de la bipolarité: étude de 100 cas

    Science.gov (United States)

    Nabih, Fadoua Oueriagli; Benali, Abdesslam; Adali, Imane; Manoudi, Fatiha; Asri, Fatima

    2015-01-01

    Le trouble bipolaire (TB) est une pathologie dont la prévalence est estimée à 1-2%. Le diagnostic précoce du trouble constitue un enjeu thérapeutique majeur. L'objectif de ce travail est de déterminer les différents diagnostiques attribués aux patients bipolaires avant de recevoir le diagnostic adéquat et de préciser le délai moyen du retard diagnostique. C'est une étude descriptive transversale portant sur 100 patients atteints de TB, inclus selon les critères du DSM V, qui ont été vus en consultation ou bien hospitalisés dans le service de psychiatrie de l'hôpital Militaire Avicenne de Marrakech, durant une période de deux ans. L’âge moyen des patients était de 29,5 ans avec une prédominance masculine (80%). 40% de nos patients ont reçu au début un autre diagnostic que celui du TB et le premier diagnostic retenu était celui de l’épisode dépressif majeur dans 36% des cas, suivi de l'accès psychotique aigu dans 28% des cas, la schizophrénie dans 16,8% et le trouble de personnalité dans 10,2%. Le délai moyen du retard diagnostic était de 64 mois. 50% des patients ayant reçu un autre diagnostic avaient consulté au moins un psychiatre et 60% des patients avaient été hospitalisés au moins une fois avant le diagnostic du TB. Les errances diagnostiques du TB sont bien établies, conduisant forcément à un retard de prise en charge adéquate. Ces données devraient alerter les psychiatres pour favoriser un meilleur dépistage de la manie et de l'hypomanie qui restent les éléments clé du diagnostic du TB. PMID:26587161

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Science and Bioethics of CRISPR-Cas9 Gene Editing: An Analysis Towards Separating Facts and Fiction
.

    Science.gov (United States)

    Cribbs, Adam P; Perera, Sumeth M W

    2017-12-01

    Since its emergence in 2012, the genome editing technique known as CRISPR-Cas9 and its scientific use have rapidly expanded globally within a very short period of time. The technique consists of using an RNA guide molecule to bind to complementary DNA sequences, which simultaneously recruits the endonuclease Cas9 to introduce double-stranded breaks in the target DNA. The resulting double-stranded break is then repaired, allowing modification or removal of specific DNA bases. The technique has gained momentum in the laboratory because it is cheap, quick, and easy to use. Moreover, it is also being applied in vivo to generate more complex animal model systems. Such use of genome editing has proven to be highly effective and warrants a potential therapy for both genetic and non-genetic diseases. Although genome editing has the potential to be a transformative therapy for patients it is still in its infancy. Consequently, the legal and ethical frameworks are yet to be fully discussed and will be an increasingly important topic as the technology moves towards more contentious issues such as modification of the germline. Here, we review a number of scientific and ethical issues which may potentially influence the development of both the technology and its use in the clinical setting.

  1. Safety apparatus for serious radioactive accidents (1962); Materiel d'intervention en cas d'accident radioactif grave (1962)

    Energy Technology Data Exchange (ETDEWEB)

    Estournel, R; Rodier, J [Commissariat a l' Energie Atomique, Centre de Production de Plutonium, Marcoule (France). Centre d' Etudes Nucleaires

    1962-07-01

    In the case of a serious radioactive accident, radioactive dust and gases may be released into the atmosphere. It is therefore necessary to be able to evaluate rapidly the importance of the risk to the surrounding population, and to be able to ensure, even in the event of an evacuation of the Centre, the continuation of the radioactivity analyses and the decontamination of the personnel. For this, the Anti-radiation Protection Service at Marcoule has organised mobile detection teams and designed a mobile laboratory and a mobile shower-unit. After describing the duty of the mobile teams, the report gives a description of the apparatus which would be used at the Marcoule Centre in the case of a serious radioactive accident. The method of using this apparatus is given. (authors) [French] Lors d'un accident radioactif grave, des poussieres et des gaz radioactifs peuvent etre relaches dans l'atmosphere. II est alors indispensable d'evaluer rapidement l'importance du risque couru par les populations environnantes, et de pouvoir assurer, meme dans le cas de l'evacuation du Centre, la poursuite des analyses radioactives et la decontamination du personnel. Pour cela, le Service de Protection contre les Radiations du Centre de Marcoule a mis sur pied des equipes mobiles de detection et realise une semi-remorque laboratoire ainsi qu'une semi-remorque douches. Apres avoir defini la mission des equipes mobiles, le rapport donne la description du materiel d'intervention qui serait mis en oeuvre par le Centre de Marcoule dans le cas d'un accident radioactif grave. Il precis le mode d'utilisation de ce materiel. (auteurs)

  2. Unique Physician Identification Number (UPIN) Directory

    Data.gov (United States)

    U.S. Department of Health & Human Services — The Unique Physician Identification Number (UPIN) Directory contains selected information on physicians, doctors of Osteopathy, limited licensed practitioners and...

  3. 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.; hide

    2015-01-01

    The CRISPR (Clustered, Regularly Interspaced, Short Palindromic Repeats)/Cas9 system has revolutionized genome editing by providing unprecedented DNA-targeting specificity. Here we demonstrate that this system can be also applied in vitro to fundamental cloning steps to facilitate efficient plasmid selection for transformation and selective gene insertion into plasmid vectors by cleaving unwanted plasmid byproducts with a single-guide RNA (sgRNA)-Cas9 nuclease complex. Using fluorescent and chromogenic proteins as reporters, we demonstrate that CRISPR/Cas9 cleavage excludes multiple plasmids as well as unwanted ligation byproducts resulting in an unprecedented increase in the transformation success rate from approximately 20% to nearly 100%. Thus, this CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER) protocol is a novel, inexpensive, and convenient application to conventional molecular cloning to achieve near-perfect selective transformation.

  4. 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

  5. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing

    Science.gov (United States)

    Church, George M.; Esvelt, Kevin; Mali, Prashant

    2017-03-07

    Methods of modulating expression of a target nucleic acid in a cell are provided including use of multiple orthogonal Cas9 proteins to simultaneously and independently regulate corresponding genes or simultaneously and independently edit corresponding genes.

  6. 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

  7. [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.

  8. [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.

  9. CAS-ATLID (co-alignment sensor of ATLID instrument) thermo-structural design and performance

    Science.gov (United States)

    Moreno, Javier; Serrano, Javier; González, David; Rodríguez, Gemma; Manjón, Andrés.; Vásquez, Eloi; Carretero, Carlos; Martínez, Berta

    2017-11-01

    This paper describes the main thermo-mechanical design features and performances of the Co-Alignment Sensor (CAS) developed by LIDAX and CRISA under ESA program with AIRBUS Defence and Space as industry prime.

  10. Simple Meets Single: The Application of CRISPR/Cas9 in Haploid Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Zixi Yin

    2017-01-01

    Full Text Available The CRISPR/Cas9 system provides a powerful method for the genetic manipulation of the mammalian genome, allowing knockout of individual genes as well as the generation of genome-wide knockout cell libraries for genetic screening. However, the diploid status of most mammalian cells restricts the application of CRISPR/Cas9 in genetic screening. Mammalian haploid embryonic stem cells (haESCs have only one set of chromosomes per cell, avoiding the issue of heterozygous recessive mutations in diploid cells. Thus, the combination of haESCs and CRISPR/Cas9 facilitates the generation of genome-wide knockout cell libraries for genetic screening. Here, we review recent progress in CRISPR/Cas9 and haPSCs and discuss their applications in genetic screening.

  11. CRISPR-Cas9 Corrects Mutation in Immune Disorder, Suggesting New Therapeutic Approach | FNLCR Staging

    Science.gov (United States)

    Gene editing using the powerful new CRISPR-Cas9 system is showing promise as a tool for developing potential treatments for inherited diseases, particularly for those caused by single genetic defects. Examples of these diseases are cystic fibrosis, m

  12. Efficient CRISPR-Cas9 Gene Disruption System in Edible-Medicinal Mushroom Cordyceps militaris

    Directory of Open Access Journals (Sweden)

    Bai-Xiong Chen

    2018-06-01

    Full Text Available Cordyceps militaris is a well-known edible medicinal mushroom in East Asia that contains abundant and diverse bioactive compounds. Since traditional genome editing systems in C. militaris were inefficient and complicated, here, we show that the codon-optimized cas9, which was used with the newly reported promoter Pcmlsm3 and terminator Tcmura3, was expressed. Furthermore, with the help of the negative selection marker ura3, a CRISPR-Cas9 system that included the Cas9 DNA endonuclease, RNA presynthesized in vitro and a single-strand DNA template efficiently generated site-specific deletion and insertion. This is the first report of a CRISPR-Cas9 system in C. militaris, and it could accelerate the genome reconstruction of C. militaris to meet the need for rapid development in the fungi industry.

  13. CRISPR/Cas9-mediated genome engineering of CHO cell factories: application and perspectives

    DEFF Research Database (Denmark)

    Lee, Jae Seong; Grav, Lise Marie; Lewis, Nathan E.

    2015-01-01

    repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system enables rapid,easy and efficient engineering of mammalian genomes. It has a wide range of applications frommodification of individual genes to genome-wide screening or regulation of genes. Facile genomeediting using CRISPR/Cas9 empowers...... researchers in the CHO community to elucidate the mechanisticbasis behind high level production of proteins and product quality attributes of interest. Inthis review, we describe the basis of CRISPR/Cas9-mediated genome editing and its applicationfor development of next generation CHO cell factories while...... highlighting both future perspectivesand challenges. As one of the main drivers for the CHO systems biology era, genome engineeringwith CRISPR/Cas9 will pave the way for rational design of CHO cell factories....

  14. Genes Linked to Production of Secondary Metabolites in Talaromyces atroroseus Revealed Using CRISPR-Cas9

    DEFF Research Database (Denmark)

    Nielsen, Maria Lund; Petersen, Thomas Isbrandt; Rasmussen, Kasper Bøwig

    2017-01-01

    The full potential of fungal secondary metabolism has until recently been impeded by the lack of universal genetic tools for most species. However, the emergence of several CRISPR-Cas9-based genome editing systems adapted for several genera of filamentous fungi have now opened the doors for future...... efforts in discovery of novel natural products and elucidation and engineering of their biosynthetic pathways in fungi where no genetic tools are in place. So far, most studies have focused on demonstrating the performance of CRISPR-Cas9 in various fungal model species, and recently we presented...... a versatile CRISPR-Cas9 system that can be successfully applied in several diverse Aspergillus species. Here we take it one step further and show that our system can be used also in a phylogenetically distinct and largely unexplored species from the genus of Talaromyces. Specifically, we exploit CRISPR-Cas9...

  15. 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...

  16. Genome Editing in Escherichia coli with Cas9 and synthetic CRISPRs

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Ze; Richardson, Sarah; Robinson, David; Deutsch, Samuel; Cheng, Jan-Fang

    2014-03-14

    Recently, the Cas9-CRISPR system has proven to be a useful tool for genome editing in eukaryotes, which repair the double stranded breaks made by Cas9 with non-homologous end joining or homologous recombination. Escherichia coli lacks non-homologous end joining and has a very low homologous recombination rate, effectively rendering targeted Cas9 activity lethal. We have developed a heat curable, serializable, plasmid based system for selectionless Cas9 editing in arbitrary E. coli strains that uses synthetic CRISPRs for targeting and -red to effect repairs of double stranded breaks. We have demonstrated insertions, substitutions, and multi-target deletions with our system, which we have tested in several strains.

  17. PEG capped CaS nanoparticles synthesized by wet chemical co-precipitation method

    Science.gov (United States)

    Rekha, S.; Anila, E. I.

    2018-04-01

    Calcium sulfide (CaS) nanoparticles capped with polyethyleneglycol (PEG) were synthesized using wet chemical co-precipitation method. The structural and optical properties of the prepared sample were studied by X-ray diffractogram (XRD), transmission electron microscopy (TEM), diffuse reflectance spectrum (DRS) and photoluminescence (PL) spectrum. The structure of CaS nanoparticles is cubic as demonstrated by the X-ray powder diffraction (XRD) and selected area electron diffraction (SAED) analysis. TEMimage revealed the spherical morphology of the particles with diameter in the range 15-20 nm. The optical band gap of the prepared sample was determined from the DRS and its value was found to be 4.1 eV. The PL studies showed that the relative intensity of the PEG capped CaS nanoparticles was higher than that of uncapped CaS nanoparticles. The presence of various functional groups in the capped samples were examined by Fourier Transform Infrared (FTIR) spectroscopy.

  18. Method of inhibiting plant virus pathogen infections by crispr/cas9-mediated interference

    KAUST Repository

    Mahfouz, Magdy M.; Ali, Zahir

    2016-01-01

    A genetically modified tobacco plant or tomato plant resistant to at least one pathogenic geminiviridae virus species is provided. The plant comprises a heterologous CRISPR/Cas9 system and at least one heterologous nucleotide sequence

  19. Incidence of Type II CRISPR1-Cas Systems in Enterococcus Is Species-Dependent.

    Directory of Open Access Journals (Sweden)

    Casandra Lyons

    Full Text Available CRISPR-Cas systems, which obstruct both viral infection and incorporation of mobile genetic elements by horizontal transfer, are a specific immune response common to prokaryotes. Antiviral protection by CRISPR-Cas comes at a cost, as horizontally-acquired genes may increase fitness and provide rapid adaptation to habitat change. To date, investigations into the prevalence of CRISPR have primarily focused on pathogenic and clinical bacteria, while less is known about CRISPR dynamics in commensal and environmental species. We designed PCR primers and coupled these with DNA sequencing of products to detect and characterize the presence of cas1, a universal CRISPR-associated gene and proxy for the Type II CRISPR1-Cas system, in environmental and non-clinical Enterococcus isolates. CRISPR1-cas1 was detected in approximately 33% of the 275 strains examined, and differences in CRISPR1 carriage between species was significant. Incidence of cas1 in E. hirae was 73%, nearly three times that of E. faecalis (23.6% and 10 times more frequent than in E. durans (7.1%. Also, this is the first report of CRISPR1 presence in E. durans, as well as in the plant-associated species E. casseliflavus and E. sulfureus. Significant differences in CRISPR1-cas1 incidence among Enterococcus species support the hypothesis that there is a tradeoff between protection and adaptability. The differences in the habitats of enterococcal species may exert varying selective pressure that results in a species-dependent distribution of CRISPR-Cas systems.

  20. CRISPR-Cas Adaptive Immune Systems of the Sulfolobales: Unravelling Their Complexity and Diversity

    Directory of Open Access Journals (Sweden)

    Roger A. Garrett

    2015-03-01

    Full Text Available The Sulfolobales have provided good model organisms for studying CRISPR-Cas systems of the crenarchaeal kingdom of the archaea. These organisms are infected by a wide range of exceptional archaea-specific viruses and conjugative plasmids, and their CRISPR-Cas systems generally exhibit extensive structural and functional diversity. They carry large and multiple CRISPR loci and often multiple copies of diverse Type I and Type III interference modules as well as more homogeneous adaptation modules. These acidothermophilic organisms have recently provided seminal insights into both the adaptation process, the diverse modes of interference, and their modes of regulation. The functions of the adaptation and interference modules tend to be loosely coupled and the stringency of the crRNA-DNA sequence matching during DNA interference is relatively low, in contrast to some more streamlined CRISPR-Cas systems of bacteria. Despite this, there is evidence for a complex and differential regulation of expression of the diverse functional modules in response to viral infection. Recent work also supports critical roles for non-core Cas proteins, especially during Type III-directed interference, and this is consistent with these proteins tending to coevolve with core Cas proteins. Various novel aspects of CRISPR-Cas systems of the Sulfolobales are considered including an alternative spacer acquisition mechanism, reversible spacer acquisition, the formation and significance of antisense CRISPR RNAs, and a novel mechanism for avoidance of CRISPR-Cas defense. Finally, questions regarding the basis for the complexity, diversity, and apparent redundancy, of the intracellular CRISPR-Cas systems are discussed.

  1. CRISPR-Cas9 for in vivo Gene Therapy: Promise and Hurdles

    Directory of Open Access Journals (Sweden)

    Wei-Jing Dai

    2016-01-01

    Full Text Available Owing to its easy-to-use and multiplexing nature, the genome editing tool CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats (CRISPR associated nuclease 9 is revolutionizing many areas of medical research and one of the most amazing areas is its gene therapy potentials. Previous explorations into the therapeutic potentials of CRISPR-Cas9 were mainly conducted in vitro or in animal germlines, the translatability of which, however, is either limited (to tissues with adult stem cells amenable to culture and manipulation or currently impermissible (due to ethic concerns. Recently, important progresses have been made on this regard. Several studies have demonstrated the ability of CRISPR-Cas9 for in vivo gene therapy in adult rodent models of human genetic diseases delivered by methods that are potentially translatable to human use. Although these recent advances represent a significant step forward to the eventual application of CRISPR-Cas9 to the clinic, there are still many hurdles to overcome, such as the off-target effects of CRISPR-Cas9, efficacy of homology-directed repair, fitness of edited cells, immunogenicity of therapeutic CRISPR-Cas9 components, as well as efficiency, specificity, and translatability of in vivo delivery methods. In this article, we introduce the mechanisms and merits of CRISPR-Cas9 in genome editing, briefly retrospect the applications of CRISPR-Cas9 in gene therapy explorations and highlight recent advances, later we discuss in detail the challenges lying ahead in the way of its translatability, propose possible solutions, and future research directions.

  2. CRISPR-Cas Systems in Bacteroides fragilis, an Important Pathobiont in the Human Gut Microbiome

    OpenAIRE

    Tajkarimi, Mehrdad; Wexler, Hannah M.

    2017-01-01

    Background: While CRISPR-Cas systems have been identified in bacteria from a wide variety of ecological niches, there are no studies to describe CRISPR-Cas elements in Bacteroides species, the most prevalent anaerobic bacteria in the lower intestinal tract. Microbes of the genus Bacteroides make up ~25% of the total gut microbiome. Bacteroides fragilis comprises only 2% of the total Bacteroides in the gut, yet causes of >70% of Bacteroides infections. The factors causing it to transition from...

  3. Introduction to the 'CAS' nuclear propulsion plant for ships: specific safety options

    International Nuclear Information System (INIS)

    Verdeau, J.J.; Baujat, J.

    1978-01-01

    After a brief review of the development of nuclear propulsion in FRANCE (Land Based Prototype PAT 1964 - Navy nuclear ships - Advanced Nuclear Boiler Prototype CAP 1975 and now the CAS nuclear plant), the specific safety options of CAS are presented: cold, compartmented fuel (plates); reduced flow during LOCA; permanent cooling of fuel during LOCA; pressurized, entirely passive containment; no control rod ejection and possibility of temporary storage of spent fuel on board [fr

  4. Le syndrome de Pepper: à propos de deux cas observés au Centre ...

    African Journals Online (AJOL)

    Le syndrome de Pepper est une forme métastatique hépatique du neuroblastome. C'est une entité spécifique du nourrisson de moins de six mois qui a la particularité de pouvoir régresser de façon spontanée avec un pronostic favorable dans 80% des cas. A cause de sa rareté, nous rapportons deux cas du syndrome de ...

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

    Science.gov (United States)

    Pliatsika, Venetia; Rigoutsos, Isidore

    2015-01-29

    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 protospacer adjacent motif (PAM). Care needs to be exercised during gRNA design to avoid unintended ("off-target") interactions. We designed and implemented the Off-Spotter algorithm to assist with the design of optimal gRNAs. When presented with a candidate gRNA sequence and a PAM, Off-Spotter quickly and exhaustively identifies all genomic sites that satisfy the PAM constraint and are identical or nearly-identical to the provided gRNA. Off-Spotter achieves its extreme performance through purely algorithmic means and not through hardware accelerators such as graphical processing units (GPUs). Off-Spotter also allows the user to identify on-the-fly how many and which nucleotides of the gRNA comprise the "seed". Off-Spotter's output includes a histogram showing the number of potential off-targets as a function of the number of mismatches. The output also includes for each potential off-target the site's genomic location, a human genome browser hyperlink to the corresponding location, genomic annotation in the vicinity of the off-target, GC content, etc. Off-Spotter is very fast and flexible and can help in the design of optimal gRNAs by providing several PAM choices, a run-time definition of the seed and of the allowed number of mismatches, and a flexible output interface that allows sorting of the results, optional viewing/hiding of columns, etc. A key element of Off-Spotter is that it does not have a rigid definition of the seed: instead, the user can declare both the seed's location and extent

  6. A Biophysical Model of CRISPR/Cas9 Activity for Rational Design of Genome Editing and Gene Regulation

    Science.gov (United States)

    Farasat, Iman; Salis, Howard M.

    2016-01-01

    The ability to precisely modify genomes and regulate specific genes will greatly accelerate several medical and engineering applications. The CRISPR/Cas9 (Type II) system binds and cuts DNA using guide RNAs, though the variables that control its on-target and off-target activity remain poorly characterized. Here, we develop and parameterize a system-wide biophysical model of Cas9-based genome editing and gene regulation to predict how changing guide RNA sequences, DNA superhelical densities, Cas9 and crRNA expression levels, organisms and growth conditions, and experimental conditions collectively control the dynamics of dCas9-based binding and Cas9-based cleavage at all DNA sites with both canonical and non-canonical PAMs. We combine statistical thermodynamics and kinetics to model Cas9:crRNA complex formation, diffusion, site selection, reversible R-loop formation, and cleavage, using large amounts of structural, biochemical, expression, and next-generation sequencing data to determine kinetic parameters and develop free energy models. Our results identify DNA supercoiling as a novel mechanism controlling Cas9 binding. Using the model, we predict Cas9 off-target binding frequencies across the lambdaphage and human genomes, and explain why Cas9’s off-target activity can be so high. With this improved understanding, we propose several rules for designing experiments for minimizing off-target activity. We also discuss the implications for engineering dCas9-based genetic circuits. PMID:26824432

  7. RNA virus interference via CRISPR/Cas13a system in plants

    KAUST Repository

    Aman, Rashid

    2017-11-04

    CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants. CRISPR/Cas13a produced interference against green fluorescent protein (GFP) expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. crRNAs targeting the HC-Pro and GFP sequences exhibited better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs. Our data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses, and for other RNA manipulations in plants.

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

    Science.gov (United States)

    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.

  9. Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations

    Science.gov (United States)

    Zuo, Zhicheng; Liu, Jin

    2016-11-01

    The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome editing. Despite recent advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg2+ ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg2+ bound. The simulation results showed that binding of two Mg2+ ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we demonstrated with our simulations that Cas9-catalyzed DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  12. New applications of CRISPR/Cas9 system on mutant DNA detection.

    Science.gov (United States)

    Jia, Chenqiang; Huai, Cong; Ding, Jiaqi; Hu, Lingna; Su, Bo; Chen, Hongyan; Lu, Daru

    2018-01-30

    The detection of mutant DNA is critical for precision medicine, but low-frequency DNA mutation is very hard to be determined. CRISPR/Cas9 is a robust tool for in vivo gene editing, and shows the potential for precise in vitro DNA cleavage. Here we developed a DNA mutation detection system based on CRISPR/Cas9 that can detect gene mutation efficiently even in a low-frequency condition. The system of CRISPR/Cas9 cleavage in vitro showed a high accuracy similar to traditional T7 endonuclease I (T7E1) assay in estimating mutant DNA proportion in the condition of normal frequency. The technology was further used for low-frequency mutant DNA detection of EGFR and HBB somatic mutations. To the end, Cas9 was employed to cleave the wild-type (WT) DNA and to enrich the mutant DNA. Using amplified fragment length polymorphism analysis (AFLPA) and Sanger sequencing, we assessed the sensitivity of CRISPR/Cas9 cleavage-based PCR, in which mutations at 1%-10% could be enriched and detected. When combined with blocker PCR, its sensitivity reached up to 0.1%. Our results suggested that this new application of CRISPR/Cas9 system is a robust and potential method for heterogeneous specimens in the clinical diagnosis and treatment management. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Applications of the CRISPR-Cas9 system in kidney research.

    Science.gov (United States)

    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.

  14. Active Intracellular Delivery of a Cas9/sgRNA Complex Using Ultrasound-Propelled Nanomotors.

    Science.gov (United States)

    Hansen-Bruhn, Malthe; de Ávila, Berta Esteban-Fernández; Beltrán-Gastélum, Mara; Zhao, Jing; Ramírez-Herrera, Doris E; Angsantikul, Pavimol; Vesterager Gothelf, Kurt; Zhang, Liangfang; Wang, Joseph

    2018-03-01

    Direct and rapid intracellular delivery of a functional Cas9/sgRNA complex using ultrasound-powered nanomotors is reported. The Cas9/sgRNA complex is loaded onto the nanomotor surface through a reversible disulfide linkage. A 5 min ultrasound treatment enables the Cas9/sgRNA-loaded nanomotors to directly penetrate through the plasma membrane of GFP-expressing B16F10 cells. The Cas9/sgRNA is released inside the cells to achieve highly effective GFP gene knockout. The acoustic Cas9/sgRNA-loaded nanomotors display more than 80 % GFP knockout within 2 h of cell incubation compared to 30 % knockout using static nanowires. More impressively, the nanomotors enable highly efficient knockout with just 0.6 nm of the Cas9/sgRNA complex. This nanomotor-based intracellular delivery method thus offers an attractive route to overcome physiological barriers for intracellular delivery of functional proteins and RNAs, thus indicating considerable promise for highly efficient therapeutic applications. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET.

    Science.gov (United States)

    Yang, Mengyi; Peng, Sijia; Sun, Ruirui; Lin, Jingdi; Wang, Nan; Chen, Chunlai

    2018-01-09

    Off-target binding and cleavage by Cas9 pose major challenges in its application. How the conformational dynamics of Cas9 govern its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single-molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms spontaneously transits among three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We also uncovered surprising long-range allosteric communication between the HNH domain and the RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated that a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the PAM-distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  16. A lentivirus-free inducible CRISPR-Cas9 system for efficient targeting of human genes.

    Science.gov (United States)

    Bisht, Kamlesh; Grill, Sherilyn; Graniel, Jacqueline; Nandakumar, Jayakrishnan

    2017-08-01

    CRISPR-Cas9 is a cutting-edge tool for modifying genomes. The efficacy with which Cas9 recognizes its target has revolutionized the engineering of knockouts. However this efficacy complicates the knocking out of important genes in cultured cells. Unedited cells holding a survival advantage within an edited population can confound the knockout phenotype. Here we develop a HeLa-based system that overcomes this limitation, incorporating several attractive features. First, we use Flp-recombinase to generate clones stably integrated for Cas9 and guide RNAs, eliminating the possibility of unedited cells. Second, Cas9 can be induced uniformly in the clonal cultures using doxycycline to measure the knockout phenotype. Third, two genes can be simultaneously knocked out using this approach. Finally, by not involving lentiviruses, our method is appealing to a broad research audience. Using this methodology we generated an inducible AGO2-knockout cell line showing normal RNA interference in the absence of doxycycline. Upon induction of Cas9, the AGO2 locus was cleaved, the AGO2 protein was depleted, and RNA interference was compromised. In addition to generating inducible knockouts, our technology can be adapted to improve other applications of Cas9, including transcriptional/epigenetic modulation and visualization of cellular DNA loci. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  17. CRISPR-Cas9 technology and its application in haematological disorders

    Science.gov (United States)

    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

  18. 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.

  19. RNA virus interference via CRISPR/Cas13a system in plants

    KAUST Repository

    Aman, Rashid

    2018-01-04

    CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single-stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants.CRISPR/Cas13a produces interference against green fluorescent protein (GFP)-expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. CRISPR RNA (crRNAs) targeting the HC-Pro and GFP sequences exhibit better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs.Our data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses and for other RNA manipulations in plants.

  20. Harnessing CRISPR/Cas systems for programmable transcriptional and post-transcriptional regulation

    KAUST Repository

    Mahas, Ahmed

    2017-11-29

    Genome editing has enabled broad advances and novel approaches in studies of gene function and structure; now, emerging methods aim to precisely engineer post-transcriptional processes. Developing precise, efficient molecular tools to alter the transcriptome holds great promise for biotechnology and synthetic biology applications. Different approaches have been employed for targeted degradation of RNA species in eukaryotes, but they lack programmability and versatility, thereby limiting their utility for diverse applications. The CRISPR/Cas9 system has been harnessed for genome editing in many eukaryotic species and, using a catalytically inactive Cas9 variant, the CRISPR/dCas9 system has been repurposed for transcriptional regulation. Recent studies have used other CRISPR/Cas systems for targeted RNA degradation and RNA-based manipulations. For example, Cas13a, a Type VI-A endonuclease, has been identified as an RNA-guided RNA ribonuclease and used for manipulation of RNA. Here, we discuss different modalities for targeted RNA interference with an emphasis on the potential applications of CRISPR/Cas systems as programmable transcriptional regulators for broad uses, including functional biology, biotechnology, and synthetic biology applications.