Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells.

Nat Biotechnol
Authors
Keywords
Abstract

Bacterial type II CRISPR-Cas9 systems have been widely adapted for RNA-guided genome editing and transcription regulation in eukaryotic cells, yet their in vivo target specificity is poorly understood. Here we mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). Each of the four sgRNAs we tested targets dCas9 to between tens and thousands of genomic sites, frequently characterized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility decreases dCas9 binding to other sites with matching seed sequences; thus 70% of off-target sites are associated with genes. Targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site mutated above background levels. We propose a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage.

Year of Publication
2014
Journal
Nat Biotechnol
Volume
32
Issue
7
Pages
670-6
Date Published
2014 Jul
ISSN
1546-1696
URL
DOI
10.1038/nbt.2889
PubMed ID
24752079
PubMed Central ID
PMC4145672
Links
Grant list
T32 GM007287 / GM / NIGMS NIH HHS / United States
R01-CA133404 / CA / NCI NIH HHS / United States
P01 CA042063 / CA / NCI NIH HHS / United States
P30 CA014051 / CA / NCI NIH HHS / United States
P30-CA14051 / CA / NCI NIH HHS / United States
R37 HD045022 / HD / NICHD NIH HHS / United States
R37 GM034277 / GM / NIGMS NIH HHS / United States
R01 CA133404 / CA / NCI NIH HHS / United States
DP1 MH100706 / MH / NIMH NIH HHS / United States
R01 GM034277 / GM / NIGMS NIH HHS / United States
Howard Hughes Medical Institute / United States
R01 DK097768 / DK / NIDDK NIH HHS / United States
P01-CA42063 / CA / NCI NIH HHS / United States
1DP1-MH100706 / DP / NCCDPHP CDC HHS / United States
R01-GM34277 / GM / NIGMS NIH HHS / United States