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      CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens

      research-article
      1 , 2
      Nucleic Acids Research
      Oxford University Press

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          Abstract

          CRISPOR.org is a web tool for genome editing experiments with the CRISPR–Cas9 system. It finds guide RNAs in an input sequence and ranks them according to different scores that evaluate potential off-targets in the genome of interest and predict on-target activity. The list of genomes is continuously expanded, with more 150 genomes added in the last two years. CRISPOR tries to provide a comprehensive solution from selection, cloning and expression of guide RNA as well as providing primers needed for testing guide activity and potential off-targets. Recent developments include batch design for genome-wide CRISPR and saturation screens, creating custom oligonucleotides for guide cloning and the design of next generation sequencing primers to test for off-target mutations. CRISPOR is available from http://crispor.org, including the full source code of the website and a stand-alone, command-line version.

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          Most cited references14

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          Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9

          CRISPR-Cas9-based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), Cas9 can be reprogrammed to target different sites in the genome with relative ease, but the on-target activity and off-target effects of individual sgRNAs can vary widely. Here, we use recently-devised sgRNA design rules to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results. Additionally, we profile the off-target activity of thousands of sgRNAs and develop a metric to predict off-target sites. We incorporate these findings from large-scale, empirical data to improve our computational design rules and create optimized sgRNA libraries that maximize on-target activity and minimize off-target effects to enable more effective and efficient genetic screens and genome engineering.
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            Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.

            The microbial adaptive immune system CRISPR mediates defense against foreign genetic elements through two classes of RNA-guided nuclease effectors. Class 1 effectors utilize multi-protein complexes, whereas class 2 effectors rely on single-component effector proteins such as the well-characterized Cas9. Here, we report characterization of Cpf1, a putative class 2 CRISPR effector. We demonstrate that Cpf1 mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif. Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, we identified two candidate enzymes from Acidaminococcus and Lachnospiraceae, with efficient genome-editing activity in human cells. Identifying this mechanism of interference broadens our understanding of CRISPR-Cas systems and advances their genome editing applications.
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              Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases

              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@snu.ac.kr or jskim01@snu.ac.kr
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                Author and article information

                Journal
                Nucleic Acids Res
                Nucleic Acids Res
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                02 July 2018
                14 May 2018
                14 May 2018
                : 46
                : Web Server issue
                : W242-W245
                Affiliations
                [1 ]Muséum national d’Histoire naturelle, INSERM U1154, CNRS UMR 7196, Sorbonne Universités, 43 rue Cuvier, Paris F-75231, France
                [2 ]Genomics Institute, University of California at Santa Cruz, 1156 High Street, MS CBSE, Santa Cruz, CA 95060, USA
                Author notes
                To whom correspondence should be addressed. Tel: +1 831 295 0653; Fax: +1 831 459 1809; Email: max@ 123456soe.ucsc.edu
                Article
                gky354
                10.1093/nar/gky354
                6030908
                29762716
                ea3d0b10-18a2-4131-9119-f73ffb87213a
                © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 23 April 2018
                : 13 April 2018
                : 04 March 2018
                Page count
                Pages: 4
                Funding
                Funded by: California Institute of Regenerative Medicine 10.13039/100007557
                Award ID: GC1R-06673-C
                Funded by: National Institutes of Health 10.13039/100000002
                Award ID: 5U54HG007990-04
                Funded by: National Human Genome Research Institute 10.13039/100000051
                Award ID: 2U41HG002371-18
                Funded by: Chan-Zuckerberg
                Award ID: 182809
                Categories
                Web Server Issue

                Genetics
                Genetics

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