Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells

Nat Methods. 2015 Mar;12(3):237-43, 1 p following 243. doi: 10.1038/nmeth.3284. Epub 2015 Feb 9.


Although RNA-guided genome editing via the CRISPR-Cas9 system is now widely used in biomedical research, genome-wide target specificities of Cas9 nucleases remain controversial. Here we present Digenome-seq, in vitro Cas9-digested whole-genome sequencing, to profile genome-wide Cas9 off-target effects in human cells. This in vitro digest yields sequence reads with the same 5' ends at cleavage sites that can be computationally identified. We validated off-target sites at which insertions or deletions were induced with frequencies below 0.1%, near the detection limit of targeted deep sequencing. We also showed that Cas9 nucleases can be highly specific, inducing off-target mutations at merely several, rather than thousands of, sites in the entire genome and that Cas9 off-target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs. Digenome-seq is a robust, sensitive, unbiased and cost-effective method for profiling genome-wide off-target effects of programmable nucleases including Cas9.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Endonucleases / genetics
  • Endonucleases / metabolism
  • Genome, Human
  • Haploidy
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Limit of Detection
  • Mutation
  • RNA, Guide / genetics
  • Reproducibility of Results
  • Sensitivity and Specificity


  • RNA, Guide
  • Endonucleases

Associated data

  • SRA/SRX743626