CRISPR-Cas9 is an RNA-guided DNA endonuclease that has exciting genome editing applications. Cas9 cleaves DNA via its RuvC and HNH nucleases after recognizing the downstream protospacer adjacent motif (PAM) by an intriguing allosteric mechanism that is suggested to control enzymatic activity through flexibility of HNH. Structural biology has provided insight into the conformational changes that occur during DNA binding and enzyme activation, however, the allosteric mechanism by which PAM binding is detected by the nucleases from 30 Å away is unknown, as is the way in which nuclease domains with active sites 20 Å apart synchronize their function. Understanding this allosteric pathway at atomic resolution can unravel this mystery and lead to a more efficient and tunable Cas9. At 150 kDa, Cas9 is very large for NMR studies, so in order to probe dynamic allostery in Cas9 with NMR, we engineered a novel construct of HNH that maintains its wild-type fold in solution (Fig 1A). Here, we report the first integrated NMR, crystallgraphic, and computational study of allosteric signaling in the HNH nuclease (Fig. 1B). We probed picosecond-to-millisecond conformational dynamics of HNH and identified a dynamic pathway that enables information transfer across HNH (Fig. 1C), spanning its interfaces with the RuvC nuclease and DNA recognition lobe. We propose that this dynamic network is a potential route of allosteric signal propagation that coordinates cleavage of double-stranded DNA and have expanded this work to other Cas9 subdomains implicated in its allosteric mechanism, to further unravel the structural and dynamic determinants of Cas9 allostery.

Allosteric motions of the CRISPR-Cas9 endonuclease probed by NMR and molecular dynamics

ACS Fall 2021

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.