Characterize the Pre-catalytic State of CRISPR/Cas9

Purpose: CRISPR-Cas9 has been widely used as a gene-editing tool, but its catalytic mechanism remains elusive. Cas9 has two catalytic domains, HNH domain and RuvC domain. In our previous study, we have successfully identified the pre-catalytic state of the HNH domain. The purpose of this study is to identify the pre-catalytic state of the RuvC domain, which may provide an understanding of the Cas9 catalytic mechanism and strategies for Cas9 engineering. Methods: Here we use molecular dynamic simulations to discover the pre-catalytic state of Cas9. The initial structure was obtained from our previous simulations, where the HNH domain was in the pre-catalytic state. We placed two Mg2+ ions and non-target DNA strand at the RuvC domain and performed molecular dynamics simulations to capture the pre-catalytic state of the RuvC domain. Results: Our molecular dynamics simulations revealed a pre-catalytic state of Cas9 that both target DNA strand and non-target DNA strand are posed to be catalyzed by Cas9. Conclusions: In this study, we identified the first atomic-level structure of CRISPR/Cas9 with the pre-catalytic state in both catalytic domains.