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ABSTRACT 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. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution

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subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS AN ALPHA-HELICAL LID GUIDES THE TARGET DNA TOWARD CATALYSIS IN CRISPR-CAS12A Article Open

access 17 February 2024 MECHANISMS FOR TARGET RECOGNITION AND CLEAVAGE BY THE CAS12I RNA-GUIDED ENDONUCLEASE Article 07 September 2020 MECHANISTIC INSIGHTS INTO THE R-LOOP FORMATION AND

CLEAVAGE IN CRISPR-CAS12I1 Article Open access 09 June 2021 DATA AVAILABILITY 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

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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.). AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Department of Bioengineering, University of California Riverside, Riverside, CA, USA Łukasz Nierzwicki, Rohaine V. Hsu, Mohd Ahsan, Pablo R. Arantes & Giulia Palermo *

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, USA Kyle W. East, Erin Skeens & George P. Lisi * Department of Biochemistry, University

of Zürich, Zurich, Switzerland Jonas M. Binz, Martin Pacesa & Martin Jinek * Department of Chemistry, University of California Riverside, Riverside, CA, USA Giulia Palermo Authors *

Łukasz Nierzwicki View author publications You can also search for this author inPubMed Google Scholar * Kyle W. East View author publications You can also search for this author inPubMed 

Google Scholar * Jonas M. Binz View author publications You can also search for this author inPubMed Google Scholar * Rohaine V. Hsu View author publications You can also search for this

author inPubMed Google Scholar * Mohd Ahsan View author publications You can also search for this author inPubMed Google Scholar * Pablo R. Arantes View author publications You can also

search for this author inPubMed Google Scholar * Erin Skeens View author publications You can also search for this author inPubMed Google Scholar * Martin Pacesa View author publications You

can also search for this author inPubMed Google Scholar * Martin Jinek View author publications You can also search for this author inPubMed Google Scholar * George P. Lisi View author

publications You can also search for this author inPubMed Google Scholar * Giulia Palermo View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS

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. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing

interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Catalysis_ thanks Quanjiang Ji, Priyadarshi Satpati, Jeong-Yong Suh and the other, anonymous, reviewer(s) for their contribution to the

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SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Methods, Discussion, Figs. 1–29 and Tables 1 and 2. REPORTING SUMMARY 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. RIGHTS AND PERMISSIONS Springer Nature or its licensor holds exclusive rights to

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terms of such publishing agreement and applicable law. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 06 February 2022 *

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