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Nat Biotechnol. 2014 Jul;32(7):677-83. doi: 10.1038/nbt.2916. Epub 2014 May 18.

Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease.

Author information

1
1] School of Medicine, Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia, USA. [2].
2
1] School of Medicine, Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia, USA. [2] Department of Biology, Pamukkale University, Denizli, Turkey. [3].
3
1] School of Medicine, Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia, USA. [2] Department of Computer Science, University of Virginia, Charlottesville, Virginia, USA.
4
School of Medicine, Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia, USA.

Abstract

RNA-guided genome editing with the CRISPR-Cas9 system has great potential for basic and clinical research, but the determinants of targeting specificity and the extent of off-target cleavage remain insufficiently understood. Using chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we mapped genome-wide binding sites of catalytically inactive Cas9 (dCas9) in HEK293T cells, in combination with 12 different single guide RNAs (sgRNAs). The number of off-target sites bound by dCas9 varied from ∼10 to >1,000 depending on the sgRNA. Analysis of off-target binding sites showed the importance of the PAM-proximal region of the sgRNA guiding sequence and that dCas9 binding sites are enriched in open chromatin regions. When targeted with catalytically active Cas9, some off-target binding sites had indels above background levels in a region around the ChIP-seq peak, but generally at lower rates than the on-target sites. Our results elucidate major determinants of Cas9 targeting, and we show that ChIP-seq allows unbiased detection of Cas9 binding sites genome-wide.

Comment in

PMID:
24837660
DOI:
10.1038/nbt.2916
[Indexed for MEDLINE]

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