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      Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases

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      1 , 2 , * , 3 , , 1 , 2 , *
      Bioinformatics
      Oxford University Press

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          Abstract

          Summary: The Type II clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system is an adaptive immune response in prokaryotes, protecting host cells against invading phages or plasmids by cleaving these foreign DNA species in a targeted manner. CRISPR/Cas-derived RNA-guided engineered nucleases (RGENs) enable genome editing in cultured cells, animals and plants, but are limited by off-target mutations. Here, we present a novel algorithm termed Cas-OFFinder that searches for potential off-target sites in a given genome or user-defined sequences. Unlike other algorithms currently available for identification of RGEN off-target sites, Cas-OFFinder is not limited by the number of mismatches and allows variations in protospacer-adjacent motif sequences recognized by Cas9, the essential protein component in RGENs. Cas-OFFinder is available as a command-line program or accessible via our website.

          Availability and implementation: Cas-OFFinder free access at http://www.rgenome.net/cas-offinder.

          Contact: baesau@ 123456snu.ac.kr or jskim01@ 123456snu.ac.kr

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          Most cited references7

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          A TALE nuclease architecture for efficient genome editing.

          Nucleases that cleave unique genomic sequences in living cells can be used for targeted gene editing and mutagenesis. Here we develop a strategy for generating such reagents based on transcription activator-like effector (TALE) proteins from Xanthomonas. We identify TALE truncation variants that efficiently cleave DNA when linked to the catalytic domain of FokI and use these nucleases to generate discrete edits or small deletions within endogenous human NTF3 and CCR5 genes at efficiencies of up to 25%. We further show that designed TALEs can regulate endogenous mammalian genes. These studies demonstrate the effective application of designed TALE transcription factors and nucleases for the targeted regulation and modification of endogenous genes.
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            Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis.

            Genome engineering in human pluripotent stem cells (hPSCs) holds great promise for biomedical research and regenerative medicine. Recently, an RNA-guided, DNA-cleaving interference pathway from bacteria [the type II clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) pathway] has been adapted for use in eukaryotic cells, greatly facilitating genome editing. Only two CRISPR-Cas systems (from Streptococcus pyogenes and Streptococcus thermophilus), each with their own distinct targeting requirements and limitations, have been developed for genome editing thus far. Furthermore, limited information exists about homology-directed repair (HDR)-mediated gene targeting using long donor DNA templates in hPSCs with these systems. Here, using a distinct CRISPR-Cas system from Neisseria meningitidis, we demonstrate efficient targeting of an endogenous gene in three hPSC lines using HDR. The Cas9 RNA-guided endonuclease from N. meningitidis (NmCas9) recognizes a 5'-NNNNGATT-3' protospacer adjacent motif (PAM) different from those recognized by Cas9 proteins from S. pyogenes and S. thermophilus (SpCas9 and StCas9, respectively). Similar to SpCas9, NmCas9 is able to use a single-guide RNA (sgRNA) to direct its activity. Because of its distinct protospacer adjacent motif, the N. meningitidis CRISPR-Cas machinery increases the sequence contexts amenable to RNA-directed genome editing.
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              • Record: found
              • Abstract: not found
              • Article: not found

              Enhancing gene targeting with designed zinc finger nucleases.

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                Author and article information

                Journal
                Bioinformatics
                Bioinformatics
                bioinformatics
                bioinfo
                Bioinformatics
                Oxford University Press
                1367-4803
                1367-4811
                15 May 2014
                24 January 2014
                24 January 2014
                : 30
                : 10
                : 1473-1475
                Affiliations
                1National Creative Research Initiatives Center for Genome Engineering, 2Department of Chemistry and 3Department of Physics and Astronomy, Seoul National University, 599 Gwanak-ro, Seoul 151-742, South Korea
                Author notes
                *To whom correspondence should be addressed.

                Associate Editor: John Hancock

                The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors.

                Article
                btu048
                10.1093/bioinformatics/btu048
                4016707
                24463181
                c8c96d6b-2470-4a62-b1cf-2f42ff12ba4c
                © The Author 2014. Published by Oxford University Press.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

                History
                : 13 November 2013
                : 27 December 2013
                : 21 January 2014
                Page count
                Pages: 3
                Categories
                Applications Notes
                Sequence Analysis

                Bioinformatics & Computational biology
                Bioinformatics & Computational biology

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