Talk abstracts
Talk on Tuesday 04:45-05:00pm submitted by Austin Raper
Kinetic mechanism of the RNA-guided endonuclease Cas9
Austin T. Raper (OSBP), Anthony A. Stephenson (OSBP)
Abstract:
Targeted destruction or editing of DNA by the CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) endonuclease Cas9 is critical for prokaryotic adaptive immunity and genome engineering. Directed by base-pair complementarity between a unique guide-RNA and a region of DNA containing a crucial recognition element termed the protospacer adjacent motif (PAM), Cas9 is efficiently and specifically localized to DNA targets for subsequent double-strand DNA cleavage by distinct HNH and RuvC nuclease active sites. Here, we use pre-steady-state kinetic and single-molecule fluorescence experiments to unify results from previous structural and biochemical studies to clarify the enzymatic mechanism of Cas9. We determined that guide-RNA binding is consistent with an induced-fit model and results in a dramatic Cas9 conformational change. Moreover, we found that association of DNA to the Cas9-RNA binary complex kinetically limits the rate of DNA cleavage within the first enzymatic turnover and an extremely slow release of DNA products limits Cas9 to be a virtual single-turnover enzyme. In contrast, DNA cleavage from a preformed ternary complex of Cas9, guide-RNA, and DNA is rapid, with HNH cleaving before RuvC. Remarkably, even at the earliest time points (3 ms), RuvC generated two DNA cleavage products suggesting an alternative DNA cleavage site. As only a single DNA product was observed from HNH cleavage, this challenges the notion that Cas9 generates blunt-ended DNA strand breaks. Furthermore, our results indicate that DNA cleavage by HNH and RuvC is controlled by the conformational states of the individual nuclease domains, with conformational activation for HNH occurring more rapidly than for RuvC. Collectively, our work defines a minimal kinetic mechanism for DNA cleavage by Cas9 and may be useful in the quest to engineer superior Cas9 variants for gene editing.
Keywords: CRISPR, Cas9, enzyme mechanism