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      Efficient In Vivo Genome Editing Using RNA-Guided Nucleases

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          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          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 26

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          Multiplex genome engineering using CRISPR/Cas systems.

          Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
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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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                Author and article information

                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
                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

                Article
                NIHMS434949
                10.1038/nbt.2501
                3686313
                23360964

                Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

                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
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                Biotechnology

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