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Edited by Kathleen Collins, University of California, Berkeley, CA, and accepted by Editorial Board Member John W. Sedat November 30, 2020 (received for review May 26, 2020)
Significance
Cas9 complexed with a programmable guide RNA has been repurposed for genome-engineering applications. Mechanistic understanding of Cas9-mediated DNA cleavage provides opportunities for rational engineering of Cas9 to improve its efficiency and specificity. Domain movements of Cas9 have been well studied before DNA cleavage but not after. In this study, we observed the HNH nuclease domain motions from DNA binding to post-DNA cleavage in real time using single-molecule fluorescence techniques. The coupling between DNA cleavage activity and Cas9 conformation demonstrated in our study provides insights and motivation for future applications.
Abstract
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.
Footnotes
- ↵1To whom correspondence may be addressed. Email: tjha{at}jhu.edu.
Author contributions: Y.W. and T.H. designed research; Y.W., J.M., H.W., and M.H.J. performed research; Y.W., D.S., and B.H. contributed new reagents/analytic tools; Y.W., J.M., H.W., D.S., and S.B. analyzed data; and Y.W., S.B., and T.H. wrote the paper.
The authors declare no competing interest.
This article is a PNAS Direct Submission. K.C. is a guest editor invited by the Editorial Board.
This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2010650118/-/DCSupplemental.
Data Availability.
Custom-written scripts for smFRET data analysis are available at GitHub (https://github.com/ywang285/smFRET_code). All other data and details about materials used are present in the article and SI Appendix.
Published under the PNAS license.
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Real-time observation of Cas9 postcatalytic domain motions
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Article Classifications
- Biological Sciences
- Biophysics and Computational Biology
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