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Efficient In Vivo Genome Editing Using RNA-Guided Nucleases

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      Abstract

      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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      Most cited references 22

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      Mfold web server for nucleic acid folding and hybridization prediction.

       M Zuker (2003)
      The abbreviated name, 'mfold web server', describes a number of closely related software applications available on the World Wide Web (WWW) for the prediction of the secondary structure of single stranded nucleic acids. The objective of this web server is to provide easy access to RNA and DNA folding and hybridization software to the scientific community at large. By making use of universally available web GUIs (Graphical User Interfaces), the server circumvents the problem of portability of this software. Detailed output, in the form of structure plots with or without reliability information, single strand frequency plots and 'energy dot plots', are available for the folding of single sequences. A variety of 'bulk' servers give less information, but in a shorter time and for up to hundreds of sequences at once. The portal for the mfold web server is http://www.bioinfo.rpi.edu/applications/mfold. This URL will be referred to as 'MFOLDROOT'.
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        A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.

        Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
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          CRISPR provides acquired resistance against viruses in prokaryotes.

          Clustered regularly interspaced short palindromic repeats (CRISPR) are a distinctive feature of the genomes of most Bacteria and Archaea and are thought to be involved in resistance to bacteriophages. We found that, after viral challenge, bacteria integrated new spacers derived from phage genomic sequences. Removal or addition of particular spacers modified the phage-resistance phenotype of the cell. Thus, CRISPR, together with associated cas genes, provided resistance against phages, and resistance specificity is determined by spacer-phage sequence similarity.
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            Author and article information

            Affiliations
            [1 ]Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA 02129 USA
            [2 ]Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA 02129 USA
            [3 ]Department of Pathology, Harvard Medical School, Boston, MA 02115 USA
            [4 ]Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA 02115 USA
            [5 ]Department of Medicine, Harvard Medical School, Boston, MA 02115 USA
            [6 ]Broad Institute, Cambridge, MA 02142 USA
            Author notes
            [†]

            These authors contributed equally to this work

            Journal
            9604648
            20305
            Nat Biotechnol
            Nat. Biotechnol.
            Nature biotechnology
            1087-0156
            1546-1696
            15 January 2013
            29 January 2013
            March 2013
            01 September 2013
            : 31
            : 3
            : 227-229
            23360964
            3686313
            10.1038/nbt.2501
            NIHMS434949

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            Funding
            Funded by: National Institute of General Medical Sciences : NIGMS
            Award ID: R01 GM088040 || GM
            Funded by: National Human Genome Research Institute : NHGRI
            Award ID: P50 HG005550 || HG
            Funded by: National Institute on Aging : NIA
            Award ID: K01 AG031300 || AG
            Funded by: National Institute of General Medical Sciences : NIGMS
            Award ID: DP1 GM105378 || GM
            Categories
            Article

            Biotechnology

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