A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture.

Abstract:

:Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided nucleases have gathered considerable excitement as a tool for genome engineering. However, questions remain about the specificity of target site recognition. Cleavage specificity is typically evaluated by low throughput assays (T7 endonuclease I assay, target amplification followed by high-throughput sequencing), which are limited to a subset of potential off-target sites. Here, we used ChIP-seq to examine genome-wide CRISPR binding specificity at gRNA-specific and gRNA-independent sites for two guide RNAs. RNA-guided Cas9 binding was highly specific to the target site while off-target binding occurred at much lower intensities. Cas9-bound regions were highly enriched in NGG sites, a sequence required for target site recognition by Streptococcus pyogenes Cas9. To determine the relationship between Cas9 binding and endonuclease activity, we applied targeted sequence capture, which allowed us to survey 1200 genomic loci simultaneously including potential off-target sites identified by ChIP-seq and by computational prediction. A high frequency of indels was observed at both target sites and one off-target site, while no cleavage activity could be detected at other ChIP-bound regions. Our results confirm the high-specificity of CRISPR endonucleases and demonstrate that sequence capture can be used as a high-throughput genome-wide approach to identify off-target activity.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

O'Geen H,Henry IM,Bhakta MS,Meckler JF,Segal DJ

doi

10.1093/nar/gkv137

subject

Has Abstract

pub_date

2015-03-31 00:00:00

pages

3389-404

issue

6

eissn

0305-1048

issn

1362-4962

pii

gkv137

journal_volume

43

pub_type

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