Protocol

Gene Correction

Volume 1114 of the series Methods in Molecular Biology pp 269-277

Date:

RNA-Guided Genome Editing of Mammalian Cells

  • Neena K. PyzochaAffiliated withBroad Institute of MIT and Harvard, Massachusetts Institute of TechnologyDepartment of Biology, Massachusetts Institute of Technology
  • , F. Ann RanAffiliated withBroad Institute of MIT and Harvard, Massachusetts Institute of Technology
  • , Patrick D. HsuAffiliated withBroad Institute of MIT and Harvard, Massachusetts Institute of Technology
  • , Feng ZhangAffiliated withBroad Institute of MIT and HarvardMcGovern Institute for Brain Research, MITDepartment of Brain and Cognitive Sciences, MITDepartment of Biological Engineering, MIT

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Abstract

The microbial CRISPR-Cas adaptive immune system can be harnessed to facilitate genome editing in eukaryotic cells (Cong L et al., Science 339, 819–823, 2013; Mali P et al., Science 339, 823–826, 2013). Here we describe a protocol for the use of the RNA-guided Cas9 nuclease from the Streptococcus pyogenes type II CRISPR system to achieve specific, scalable, and cost-efficient genome editing in mammalian cells.

Key words

CRISPR-Cas Genome editing DNA cleavage Cas9 Guide RNA PAM sequence NHEJ Gene knockout