Format

Send to

Choose Destination
Nat Biotechnol. 2014 Jul;32(7):670-6. doi: 10.1038/nbt.2889. Epub 2014 Apr 20.

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

Author information

1
1] David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [2] Computational and Systems Biology Graduate Program, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
2
1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [3].
3
1] Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [2].
4
1] David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [2] Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
5
1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [3] Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.
6
1] Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [2] Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
7
1] Computational and Systems Biology Graduate Program, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [2] Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
8
1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
9
David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
10
Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

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.

PMID:
24752079
PMCID:
PMC4145672
DOI:
10.1038/nbt.2889
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Nature Publishing Group Icon for PubMed Central
Loading ...
Support Center