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

Cem Kuscu, Sevki Arslan, Ritambhara Singh, Jeremy Thorpe, Mazhar Adli*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

571 Scopus citations


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 â ̂1/410 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.

Original languageEnglish (US)
Pages (from-to)677-683
Number of pages7
JournalNature biotechnology
Issue number7
StatePublished - Jul 2014

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Applied Microbiology and Biotechnology
  • Molecular Medicine


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