High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity

Nat Biotechnol. 2013 Sep;31(9):839-43. doi: 10.1038/nbt.2673. Epub 2013 Aug 11.


The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight guide-RNA:Cas9 complexes to cleave each of 10(12) potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two guide-RNA:Cas9 complexes. In contrast to previous models, our results show that guide-RNA:Cas9 specificity extends past a 7- to 12-base-pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific than a longer, more-active guide RNA. High concentrations of guide-RNA:Cas9 complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • DNA / genetics
  • DNA / metabolism
  • Endonucleases / genetics*
  • Endonucleases / metabolism
  • Genetic Engineering / methods*
  • Genome
  • Genomics / methods
  • HEK293 Cells
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • RNA, Small Untranslated
  • Sequence Analysis, DNA / methods*
  • Streptococcus pyogenes / enzymology
  • Streptococcus pyogenes / genetics


  • Bacterial Proteins
  • DNA
  • Endonucleases
  • RNA, Small Untranslated