At the core of the CRISPR–Cas9 genome-editing technology, the endonuclease Cas9 introduces site-specific breaks in DNA. However, precise mechanistic information to ameliorate Cas9 function is still missing. Here, multimicrosecond molecular dynamics, free energy and multiscale simulations are combined with solution NMR and DNA cleavage experiments to resolve the catalytic mechanism of target DNA cleavage. We show that the conformation of an active HNH nuclease is tightly dependent on the catalytic Mg2+, unveiling its cardinal structural role. This activated Mg2+-bound HNH is consistently described through molecular simulations, nuclear magnetic resonance (NMR) and DNA cleavage assays, revealing also that the protonation state of the catalytic H840 is strongly affected by active site mutations. Finally, ab initio quantum mechanics (density functional theory)/molecular mechanics simulations and metadynamics establish the catalytic mechanism, showing that the catalysis is activated by H840 and completed by K866, thus rationalizing DNA cleavage experiments. This information is critical to enhancing the enzymatic function of CRISPR–Cas9 towards improved genome editing.
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Atomic coordinates of the optimized computational models are available in figshare with the identifier https://doi.org/10.6084/m9.figshare.19158080. NMR resonance assignments for the HNH nuclease are available in the BMRB entry 27949. All other data are available from the authors upon reasonable request. Source data are provided with this paper.
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This material is based on work supported by the National Institute of Health (grant no. R01GM141329, to G.P.) and the National Science Foundation (grant no. CHE-1905374, to G.P.). G.P.L. is supported by the National Science Foundation (grant no. MCB-2143760). This work was also supported in part by the National Institute of Health (grant no. R01GM136815 to G.P. and G.P.L.). M.J. acknowledges support from the Swiss National Science Foundation (31003A_182567). M.J. is an International Research Scholar of the Howard Hughes Medical Institute and Vallee Scholar of the Bert L & N Kuggie Vallee Foundation. Computer time for MD has been awarded by XSEDE under grant no. TG-MCB160059 and by NERSC under grant no. M3807 (to G.P.).
The authors declare no competing interests.
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Nierzwicki, Ł., East, K.W., Binz, J.M. et al. Principles of target DNA cleavage and the role of Mg2+ in the catalysis of CRISPR–Cas9. Nat Catal 5, 912–922 (2022). https://doi.org/10.1038/s41929-022-00848-6