Genome engineering using the CRISPR-Cas9 system.

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.

Journal

Journal ID (iso-abbrev): Nat Protoc

Title: Nature protocols

Publisher: Springer Nature

ISSN (Electronic): 1750-2799

ISSN (Print): 1750-2799

Publication date (Electronic): Nov 2013

Volume: 8

Issue: 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].

Publisher Item ID: nprot.2013.143 Mid ID: NIHMS539734

DOI: 10.1038/nprot.2013.143

PMC ID: 3969860

PubMed ID: 24157548