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Principles of target DNA cleavage and the role of Mg2+ in the catalysis of CRISPR–Cas9


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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Fig. 1: Overview of the Streptococcus pyogenes (Sp) CRISPR–Cas9 system.
Fig. 2: Transition of the HNH domain from pseudo-active to active states.
Fig. 3: Chemical environment enabling the catalysis.
Fig. 4: Effect of alanine mutations on the catalytic site.
Fig. 5: Free energy profiles for phosphodiester bond cleavage.
Fig. 6: Catalytic mechanism of DNA cleavage in the HNH domain of CRISPR–Cas9.

Data availability

Atomic coordinates of the optimized computational models are available in figshare with the identifier 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.).

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Authors and Affiliations



L.N. performed molecular simulations and analysed data. K.W.E. and E.S. performed NMR experiments. J.M.B. and M.P. performed DNA cleavage experiments. P.R.A., R.V.H. and M.A. performed molecular simulations. M.J. supervised DNA cleavage experiments. G.P.L. supervised NMR experiments. G.P. conceived this research, supervised computational studies and wrote the manuscript, with critical input from all authors.

Corresponding authors

Correspondence to George P. Lisi or Giulia Palermo.

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Nature Catalysis thanks Quanjiang Ji, Priyadarshi Satpati, Jeong-Yong Suh and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Methods, Discussion, Figs. 1–29 and Tables 1 and 2.

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Source data

Source Data Fig. 3

Unprocessed gel pictures for the In vitro cleavage kinetics of Cas9 HNH mutants on a double-stranded DNA on-target substrate.

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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).

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