Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes

Mol Cell. 2015 Nov 5;60(3):398-407. doi: 10.1016/j.molcel.2015.10.030.


Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9-guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • CRISPR-Cas Systems / physiology*
  • DNA, Single-Stranded / genetics
  • DNA, Single-Stranded / metabolism*
  • Endonucleases / genetics
  • Endonucleases / metabolism*
  • RNA, Bacterial / genetics
  • RNA, Bacterial / metabolism
  • Streptococcus pyogenes / enzymology*
  • Streptococcus pyogenes / genetics


  • Bacterial Proteins
  • DNA, Single-Stranded
  • RNA, Bacterial
  • Endonucleases