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      Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR

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          Abstract

          Background

          The success of the CRISPR/Cas9 genome editing technique depends on the choice of the guide RNA sequence, which is facilitated by various websites. Despite the importance and popularity of these algorithms, it is unclear to which extent their predictions are in agreement with actual measurements.

          Results

          We conduct the first independent evaluation of CRISPR/Cas9 predictions. To this end, we collect data from eight SpCas9 off-target studies and compare them with the sites predicted by popular algorithms. We identify problems in one implementation but found that sequence-based off-target predictions are very reliable, identifying most off-targets with mutation rates superior to 0.1 %, while the number of false positives can be largely reduced with a cutoff on the off-target score. We also evaluate on-target efficiency prediction algorithms against available datasets. The correlation between the predictions and the guide activity varied considerably, especially for zebrafish. Together with novel data from our labs, we find that the optimal on-target efficiency prediction model strongly depends on whether the guide RNA is expressed from a U6 promoter or transcribed in vitro. We further demonstrate that the best predictions can significantly reduce the time spent on guide screening.

          Conclusions

          To make these guidelines easily accessible to anyone planning a CRISPR genome editing experiment, we built a new website ( http://crispor.org) that predicts off-targets and helps select and clone efficient guide sequences for more than 120 genomes using different Cas9 proteins and the eight efficiency scoring systems evaluated here.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s13059-016-1012-2) contains supplementary material, which is available to authorized users.

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          Most cited references 22

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          Efficient In Vivo Genome Editing Using RNA-Guided Nucleases

          Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have evolved in bacteria and archaea as a defense mechanism to silence foreign nucleic acids of viruses and plasmids. Recent work has shown that bacterial type II CRISPR systems can be adapted to create guide RNAs (gRNAs) capable of directing site-specific DNA cleavage by the Cas9 nuclease in vitro. Here we show that this system can function in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies comparable to those obtained using ZFNs and TALENs for the same genes. RNA-guided nucleases robustly enabled genome editing at 9 of 11 different sites tested, including two for which TALENs previously failed to induce alterations. These results demonstrate that programmable CRISPR/Cas systems provide a simple, rapid, and highly scalable method for altering genes in vivo, opening the door to using RNA-guided nucleases for genome editing in a wide range of organisms.
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            Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library.

            Identification of genes influencing a phenotype of interest is frequently achieved through genetic screening by RNA interference (RNAi) or knockouts. However, RNAi may only achieve partial depletion of gene activity, and knockout-based screens are difficult in diploid mammalian cells. Here we took advantage of the efficiency and high throughput of genome editing based on type II, clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems to introduce genome-wide targeted mutations in mouse embryonic stem cells (ESCs). We designed 87,897 guide RNAs (gRNAs) targeting 19,150 mouse protein-coding genes and used a lentiviral vector to express these gRNAs in ESCs that constitutively express Cas9. Screening the resulting ESC mutant libraries for resistance to either Clostridium septicum alpha-toxin or 6-thioguanine identified 27 known and 4 previously unknown genes implicated in these phenotypes. Our results demonstrate the potential for efficient loss-of-function screening using the CRISPR-Cas9 system.
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              Highly Specific and Efficient CRISPR/Cas9-Catalyzed Homology-Directed Repair in Drosophila

              We and others recently demonstrated that the readily programmable CRISPR/Cas9 system can be used to edit the Drosophila genome. However, most applications to date have relied on aberrant DNA repair to stochastically generate frameshifting indels and adoption has been limited by a lack of tools for efficient identification of targeted events. Here we report optimized tools and techniques for expanded application of the CRISPR/Cas9 system in Drosophila through homology-directed repair (HDR) with double-stranded DNA (dsDNA) donor templates that facilitate complex genome engineering through the precise incorporation of large DNA sequences, including screenable markers. Using these donors, we demonstrate the replacement of a gene with exogenous sequences and the generation of a conditional allele. To optimize efficiency and specificity, we generated transgenic flies that express Cas9 in the germline and directly compared HDR and off-target cleavage rates of different approaches for delivering CRISPR components. We also investigated HDR efficiency in a mutant background previously demonstrated to bias DNA repair toward HDR. Finally, we developed a web-based tool that identifies CRISPR target sites and evaluates their potential for off-target cleavage using empirically rooted rules. Overall, we have found that injection of a dsDNA donor and guide RNA-encoding plasmids into vasa-Cas9 flies yields the highest efficiency HDR and that target sites can be selected to avoid off-target mutations. Efficient and specific CRISPR/Cas9-mediated HDR opens the door to a broad array of complex genome modifications and greatly expands the utility of CRISPR technology for Drosophila research.
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                Author and article information

                Contributors
                max@soe.ucsc.edu
                jean-paul.concordet@mnhn.fr
                Journal
                Genome Biol
                Genome Biol
                Genome Biology
                BioMed Central (London )
                1474-7596
                1474-760X
                5 July 2016
                5 July 2016
                2016
                : 17
                Affiliations
                [ ]Santa Cruz Genomics Institute, MS CBSE, University of California, 1156 High Street, Santa Cruz, CA 95064 USA
                [ ]Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Square J5, Mannheim, 68159 Germany
                [ ]Institut Curie, CNRS UMR3215, INSERM U934, Paris, Cedex 05 75248 France
                [ ]CNRS UMR 7622, INSERM U1156, Sorbonne Université Paris 06, Paris, France
                [ ]Mary Lyon Centre, MRC Harwell, Didcot, UK
                [ ]TEFOR Infrastructure, Gif-sur-Yvette, France
                [ ]INSERM U1154, CNRS UMR 7196, Muséum National d’Histoire Naturelle, Paris, France
                Article
                1012
                10.1186/s13059-016-1012-2
                4934014
                27380939
                © The Author(s). 2016

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                Funding
                Funded by: National Human Genome Research Institute (US)
                Award ID: 5U41HG002371-15
                Award Recipient :
                Funded by: National Cancer Institute (US)
                Award ID: NIH/NCI 5U54HG007990-02
                Award Recipient :
                Funded by: California Institute for Regenerative Medicine (US)
                Award ID: CIRM GC1R-06673C
                Award Recipient :
                Funded by: Agence Nationale de la Recherche (FR)
                Award ID: ANR-II-INBS-0014
                Award Recipient :
                Funded by: Agence Nationale de la Recherche (FR)
                Award ID: CILIAINTHEBRAIN
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100002915, Fondation pour la Recherche Médicale;
                Award ID: FRM DEQ20140329544
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2016

                Genetics

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