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

      research-article
      Author(s): 1 , 2 , * , 3 , , 1 , 2 , *
      Publication date (Electronic):
      Journal: Bioinformatics
      Publisher: 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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          A TALE nuclease architecture for efficient genome editing.

          Jeffrey Miller, Siyuan Tan, Guijuan Qiao … (2011)
          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.

            Zhonggang Hou, Yan Yan Zhang, Nicholas E. Propson … (2013)
            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.
            Article 0 comments Cited 278 times – based on 0 reviews     Review now
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              Enhancing gene targeting with designed zinc finger nucleases.

              Marina Bibikova, Kelly Beumer, Jonathan Trautman … (2003)
              Article 0 comments Cited 246 times – based on 0 reviews     Review now
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                Author and article information

                Journal
                Journal ID (nlm-ta): Bioinformatics
                Journal ID (iso-abbrev): Bioinformatics
                Journal ID (publisher-id): bioinformatics
                Journal ID (hwp): bioinfo
                Title: Bioinformatics
                Publisher: Oxford University Press
                ISSN (Print): 1367-4803
                ISSN (Electronic): 1367-4811
                Publication date (Print): 15 May 2014
                Publication date (Electronic): 24 January 2014
                Publication date PMC-release: 24 January 2014
                Volume: 30
                Issue: 10
                Pages: 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
                Publisher ID: btu048
                DOI: 10.1093/bioinformatics/btu048
                PMC ID: 4016707
                PubMed ID: 24463181
                SO-VID: c8c96d6b-2470-4a62-b1cf-2f42ff12ba4c
                Copyright © © The Author 2014. Published by Oxford University Press.
                License:

                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
                Date received : 13 November 2013
                Date revision received : 27 December 2013
                Date accepted : 21 January 2014
                Page count
                Pages: 3
                Categories
                Subject: Applications Notes
                Subject: Sequence Analysis

                ScienceOpen disciplines: Bioinformatics & Computational biology
                Data availability:
                ScienceOpen disciplines: Bioinformatics & Computational biology

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