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      Genome engineering using the CRISPR-Cas9 system

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          Abstract

          Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.

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          Author and article information

          Journal
          Nature Protocols
          Nat Protoc
          Springer Science and Business Media LLC
          1754-2189
          1750-2799
          November 2013
          October 24 2013
          November 2013
          : 8
          : 11
          : 2281-2308
          Article
          10.1038/nprot.2013.143
          3969860
          24157548
          83168130-811c-4c34-8622-92cce7cccd70
          © 2013

          http://www.springer.com/tdm

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