Principles of DNA cleavage in CRISPR-Cas9

At the core of the CRISPR-Cas9 genome-editing technology, the endonuclease Cas9 introduces site-specific breaks in double-stranded DNA. In this system, the catalysis of target DNA cleavage by the highly flexible HNH domain is undefined, with two possible conformations whose catalytic relevance has remained unmet. Here, extensive molecular simulations - harnessing multi-microsecond molecular dynamics, quantum mechanics and free energy approaches - are combined with solution NMR and DNA cleavage assays to establish how the HNH nuclease is activated and cleaves the target DNA. We show that the conformation of the active state is critically dependent on the presence of Mg2+, unveiling its cardinal structural role in the organization of the active site. Solution NMR, DNA cleavage assays and molecular simulations of the Mg2+-bound HNH report on the formation of this active state, and consistently show that the protonation state of catalytic H840 is strongly affected by residue mutations within the active site. Finally, quantum mechanical simulations establish that DNA cleavage occurs through the identified active state, showing that the catalysis is activated by H840 and completed by K866, in line with DNA cleavage experiments. Overall, high-level computations, supported by experimental evaluation, resolve the catalytic mechanism and which of the known HNH conformations is responsible for target DNA cleavage in CRISPR-Cas9. This information helps enhancing the catalytic efficiency of Cas9 and its specificity, furthering the development of genome-editing tools.