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Nat Protoc. 2013 Nov;8(11):2281-2308. doi: 10.1038/nprot.2013.143. Epub 2013 Oct 24.

Genome engineering using the CRISPR-Cas9 system.

Ran FA#1,2,3,4,5, Hsu PD#1,2,3,4,5, Wright J1, Agarwala V1,6,7, Scott DA1,2,3,4, Zhang F1,2,3,4.

Author information

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
Program in Biophysics, Harvard University, MIT, Cambridge, Massachusetts, USA.
7
Harvard-MIT Division of Health Sciences and Technology, MIT, Cambridge, Massachusetts, USA.
#
Contributed equally

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.

PMID:
24157548
PMCID:
PMC3969860
DOI:
10.1038/nprot.2013.143
[Indexed for MEDLINE]
Free PMC Article
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