DNA targeting specificity of RNA-guided Cas9 nucleases

Nat Biotechnol. 2013 Sep;31(9):827-32. doi: 10.1038/nbt.2647. Epub 2013 Jul 21.


The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.

Publication types

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

MeSH terms

  • Bacterial Proteins / genetics
  • Base Pair Mismatch
  • Base Sequence
  • DNA / genetics*
  • Deoxyribonucleases / genetics*
  • Genetic Engineering / methods*
  • Molecular Sequence Data
  • RNA, Small Untranslated
  • Streptococcus pyogenes / genetics
  • Transcription Factors / genetics*


  • Bacterial Proteins
  • Transcription Factors
  • DNA
  • Deoxyribonucleases
  • RNA, Small Untranslated