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      Heritable and Precise Zebrafish Genome Editing Using a CRISPR-Cas System

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          Abstract

          We have previously reported a simple and customizable CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided Cas9 nuclease (RGN) system that can be used to efficiently and robustly introduce somatic indel mutations in endogenous zebrafish genes. Here we demonstrate that RGN-induced mutations are heritable, with efficiencies of germline transmission reaching as high as 100%. In addition, we extend the power of the RGN system by showing that these nucleases can be used with single-stranded oligodeoxynucleotides (ssODNs) to create precise intended sequence modifications, including single nucleotide substitutions. Finally, we describe and validate simple strategies that improve the targeting range of RGNs from 1 in every 128 basepairs (bps) of random DNA sequence to 1 in every 8 bps. Together, these advances expand the utility of the CRISPR-Cas system in the zebrafish beyond somatic indel formation to heritable and precise genome modifications.

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          Most cited references 19

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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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            Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting

            TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online.
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              FLASH Assembly of TALENs Enables High-Throughput Genome Editing

              Engineered transcription activator-like effector nucleases (TALENs) have shown promise as facile and broadly applicable genome editing tools. However, no publicly available high-throughput method for constructing TALENs has been published and large-scale assessments of the success rate and targeting range of the technology remain lacking. Here we describe the Fast Ligation-based Automatable Solid-phase High-throughput (FLASH) platform, a rapid and cost-effective method we developed to enable large-scale assembly of TALENs. We tested 48 FLASH-assembled TALEN pairs in a human cell-based EGFP reporter system and found that all 48 possessed efficient gene modification activities. We also used FLASH to assemble TALENs for 96 endogenous human genes implicated in cancer and/or epigenetic regulation and found that 84 pairs were able to efficiently introduce targeted alterations. Our results establish the robustness of TALEN technology and demonstrate that FLASH facilitates high-throughput genome editing at a scale not currently possible with engineered zinc-finger nucleases or meganucleases.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2013
                9 July 2013
                : 8
                : 7
                Affiliations
                [1 ]Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts, United States of America
                [2 ]Department of Medicine, Harvard Medical School, Boston, Massachusetts, United States of America
                [3 ]Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, United States of America
                [4 ]Department of Pathology, Harvard Medical School, Boston, Massachusetts, United States of America
                [5 ]Broad Institute, Cambridge, Massachusetts, United States of America
                Mayo Clinic, United States of America
                Author notes

                Competing Interests: One of the authors, JKJ has a financial interest in Transposagen Biopharmaceuticals. JKJs interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

                Conceived and designed the experiments: WYH YF DR MLM JDS JKJ RTP JRJY. Performed the experiments: WYH YF DR MLM PK. Analyzed the data: WYH YF DR MLM PK JDS JKJ RTP JRJY. Contributed reagents/materials/analysis tools: WYH YF DR MLM JDS. Wrote the paper: WYH YF DR MLM JDS JKJ RTP JRJY.

                Article
                PONE-D-13-13968
                10.1371/journal.pone.0068708
                3706373
                23874735

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 9
                Funding
                This work was supported by National Institutes of Health [R01 GM088040 to JKJ and RTP, NIH Director’s Pioneer Award DP1 OD006862 to JKJ, K01 AG031300 to JRJY, R01 CA140188 to JRJY]; Defense Advanced Research Projects Agency (DARPA) [W911NF-11-2-0056 to JKJ]; the Jim and Ann Orr MGH Research Scholar award (JKJ); the Charles and Ann Sanders MGH Research Scholar award (RTP); and the MGH Claflin Distinguished Scholar Award (JRJY). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Biotechnology
                Bioengineering
                Biological Systems Engineering
                Genetic Engineering
                Transgenics
                Genetics
                Genetic Mutation
                Heredity
                Model Organisms
                Animal Models
                Zebrafish
                Engineering
                Bioengineering
                Biological Systems Engineering

                Uncategorized

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