Blog
About

243
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: not found

      Genome engineering using the CRISPR-Cas9 system.

      Read this article at

      ScienceOpenPublisherPMC
      Bookmark
          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

          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.

          Related collections

          Author and article information

          Journal
          Nat Protoc
          Nature protocols
          Springer Nature
          1750-2799
          1750-2799
          Nov 2013
          : 8
          : 11
          Affiliations
          [1 ] 1] Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, Massachusetts, USA. [2] McGovern Institute for Brain Research, Cambridge, Massachusetts, USA. [3] Department of Brain and Cognitive Sciences, MIT, Cambridge, Massachusetts, USA. [4] Department of Biological Engineering, MIT, Cambridge, Massachusetts, USA. [5] Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA. [6].
          nprot.2013.143 NIHMS539734
          10.1038/nprot.2013.143
          3969860
          24157548

          Comments

          Comment on this article