Real-time observation of cas9 postcatalytic domain motions

Yanbo Wang, John Mallon, Haobo Wang, Digvijay Singh, Myung Hyun Jo, Boyang Hua, Scott Bailey, Taekjip Ha

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


CRISPR-Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease, which has become the most popular genome editing tool. Coordinated domain motions of Cas9 prior to DNA cleavage have been extensively characterized but our understanding of Cas9 conformations postcatalysis is limited. Because Cas9 can remain stably bound to the cleaved DNA for hours, its postcatalytic conformation may influence genome editing mechanisms. Here, we use single-molecule fluorescence resonance energy transfer to characterize the HNH domain motions of Cas9 that are coupled with cleavage activity of the target strand (TS) or nontarget strand (NTS) of DNA substrate. We reveal an NTS-cleavage-competent conformation following the HNH domain conformational activation. The 3′ flap generated by NTS cleavage can be rapidly digested by a 3′ to 5′ single-stranded DNA-specific exonuclease, indicating Cas9 exposes the 3′ flap for potential interaction with the DNA repair machinery. We find evidence that the HNH domain is highly flexible post-TS cleavage, explaining a recent observation that the HNH domain was not visible in a postcatalytic cryo-EM structure. Our results illuminate previously unappreciated regulatory roles of DNA cleavage activity on Cas9’s conformation and suggest possible biotechnological applications.

Original languageEnglish (US)
Article numbere2010650118
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number2
StatePublished - Jan 12 2021


  • CRISPR-cas9
  • Conformational rearrangement
  • Single molecule

ASJC Scopus subject areas

  • General


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