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

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


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Binding Sites
  • CRISPR-Cas Systems / genetics*
  • Cells, Cultured
  • Chromosome Mapping*
  • Deoxyribonuclease I / genetics*
  • Embryonic Stem Cells / physiology*
  • Gene Targeting / methods*
  • Genome / genetics*
  • HEK293 Cells
  • Humans
  • Models, Genetic*
  • Molecular Sequence Data


  • Deoxyribonuclease I

Associated data

  • GEO/GSE55887