Poster abstracts
Poster number 61 submitted by Austin Raper
Functional Insights Revealed by the Kinetic Mechanism of CRISPR/Cas9
Austin T. Raper (OSBP), Anthony A. Stephenson (OSBP)
Abstract:
The discovery of prokaryotic adaptive immunity prompted widespread use of the RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) endonuclease Cas9 for genetic engineering. However, its kinetic mechanism remains undefined, and details of DNA cleavage are poorly characterized. Here, we establish a kinetic mechanism of Streptococcus pyogenes Cas9 from guide-RNA binding through DNA cleavage and product release. Association of DNA to the binary complex of Cas9 and guide-RNA is rate-limiting during the first catalytic turnover, while DNA cleavage from a pre-formed ternary complex of Cas9, guide-RNA, and DNA is rapid. Moreover, an extremely slow release of DNA products essentially restricts Cas9 to be a single-turnover enzyme. By simultaneously measuring the contributions of the HNH and RuvC nuclease activities of Cas9 to DNA cleavage, we also uncovered the kinetic basis by which HNH conformationally regulates the RuvC cleavage activity. Together, our results provide crucial kinetic and functional details regarding Cas9 which will inform gene-editing experiments, guide future research to understand off-target DNA cleavage by Cas9, and aid in the continued development of Cas9 as a biotechnological tool.
References:
Raper, A.T., Stephenson, A.A., and Suo, Z. (2018) Functional Insights Revealed by the Kinetic Mechanism of CRISPR/Cas9. J. Am. Chem. Soc. 140, 2971-2984.
Stephenson, A.A., Raper, A.T., and Suo, Z. (2018) Bidirectional Degradation of DNA Cleavage Products Catalyzed by CRISPR/Cas9. J. Am. Chem. Soc. 140, 3743-3750.
Keywords: CRISPRCas9, gene-editing, enzyme kinetics and dynamics