UC Riverside

CRISPR-Cas9 is a bacterial immune system with exciting applications for genome editing. In spite of extensive experimental characterization, the active site chemistry of the RuvC domain-which performs DNA cleavages-has remained elusive. Its knowledge is key for structure-based engineering aimed at improving DNA cleavages. Here, we deliver an in-depth characterization by using quantum-classical (QM/MM) molecular dynamics (MD) simulations and a Gaussian accelerated MD method, coupled with bioinformatics analysis. We disclose a two-metal aided architecture in the RuvC active site, which is poised to operate DNA cleavages, in analogy with other DNA/RNA processing enzymes. The conformational dynamics of the RuvC domain further reveals that an "arginine finger" stably contacts the scissile phosphate, with the function of stabilizing the active complex. Remarkably, the formation of a catalytically competent state of the RuvC domain is only observed upon the conformational activation of the other nuclease domain of CRISPR-Cas9-i.e., the HNH domain-such allowing concerted cleavages of double stranded DNA. This structure is in agreement with the available experimental data and remarkably differs from previous models based on classical mechanics, demonstrating also that only quantum mechanical simulations can accurately describe the metal-aided active site in CRISPR-Cas9. This fully catalytic structure-in which both the HNH and RuvC domains are prone to perform DNA cleavages-constitutes a stepping-stone for understanding DNA cleavage and specificity. It calls for novel experimental verifications and offers the structural foundations for engineering efforts aimed at improving the genome editing capability of CRISPR-Cas9.