Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

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.


  1. Doudna, J. A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096 (2014).

    Article  PubMed  Google Scholar 

  2. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Jinek, M. et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 343, 1247997 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Casalino, L., Nierzwicki, Ł., Jinek, M. & Palermo, G. Catalytic mechanism of non-target DNA cleavage in CRISPR-Cas9 revealed by ab Initio molecular dynamics. ACS Catal. 10, 13596–13605 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Palermo, G., Miao, Y., Walker, R. C., Jinek, M. & McCammon, J. A. CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations. Proc. Natl Acad. Sci. USA 114, 7260–7265 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Dagdas, Y. S., Chen, J. S., Sternberg, S. H., Doudna, J. A. & Yildiz, A. A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9. Sci. Adv. 3, eaao0027 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Sternberg, S. H., LaFrance, B., Kaplan, M. & Doudna, J. A. Conformational control of DNA target cleavage by CRISPR–Cas9. Nature 527, 110–113 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Biertümpfel, C., Yang, W. & Suck, D. Crystal structure of T4 endonuclease VII resolving a Holliday junction. Nature 449, 616–620 (2007).

    Article  PubMed  Google Scholar 

  10. Zuo, Z. & Liu, J. Structure and dynamics of Cas9 HNH domain catalytic state. Sci. Rep. 7, 17271 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Anders, C., Niewoehner, O., Duerst, A. & Jinek, M. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513, 569–573 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Jiang, F. et al. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage. Science 351, 867–871 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Huai, G. et al. Structural insights into DNA cleavage activation of CRISPR-Cas9 system. Nat. Commun. 8, 1375 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  14. Zhu, X. et al. Cryo-EM structures reveal coordinated domain motions that govern DNA cleavage by Cas9. Nat. Struct. Mol. Biol. 26, 679–685 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Bravo, J. P. K. et al. Structural basis for mismatch surveillance by CRISPR–Cas9. Nature 603, 343–347 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Pacesa, M. et al. R-loop formation and conformational activation mechanisms of Cas9. Nature 609, 191–96 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Zuo, Z. et al. Structural and functional insights into the bona fide catalytic state of Streptococcus pyogenes Cas9 HNH nuclease domain. eLife 8, e46500 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Yoon, H., Zhao, L. N. & Warshel, A. Exploring the catalytic mechanism of Cas9 using information inferred from endonuclease VII. ACS Catal. 9, 1329–1336 (2019).

    Article  CAS  PubMed  Google Scholar 

  19. Zhao, L. N., Mondal, D. & Warshel, A. Exploring alternative catalytic mechanisms of the Cas9 HNH domain. Proteins 88, 260–264 (2019).

    Article  PubMed  Google Scholar 

  20. Kästner, J. Umbrella sampling. WIREs Comput. Mol. Sci. 1, 932–942 (2011).

    Article  Google Scholar 

  21. East, K. W. et al. Allosteric motions of the CRISPR–Cas9 HNH nuclease probed by NMR and molecular dynamics. J. Am. Chem. Soc. 142, 1348–1358 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Nierzwicki, Ł. et al. Enhanced specificity mutations perturb allosteric signaling in the CRISPR-Cas9 HNH endonuclease. eLife 10, e73601 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Swails, J. M. & Roitberg, A. E. Enhancing conformation and protonation state sampling of hen egg white lysozyme using pH replica exchange molecular dynamics. J. Chem. Theory Comput. 8, 4393–4404 (2012).

    Article  CAS  PubMed  Google Scholar 

  24. Hansen, A. L. & Kay, L. E. Measurement of histidine pKa values and tautomer populations in invisible protein states. Proc. Natl Acad. Sci. USA 111, 1705–1712 (2014).

    Article  Google Scholar 

  25. Shimahara, H. et al. Tautomerism of histidine 64 associated with proton transfer in catalysis of carbonic anhydrase. J. Biol. Chem. 282, 9646–9656 (2007).

    Article  CAS  PubMed  Google Scholar 

  26. Brunk, E. et al. Pushing frontiers of first-principles based computer simulations of chemical and biological systems. Chimia (Aarau) 65, 667–671 (2011).

    Article  CAS  Google Scholar 

  27. Carter, E. A., Ciccotti, G., Hynes, J. T. & Kapral, R. Constrained reaction coordinate dynamics for the simulation of rare events. Chem. Phys. Lett. 156, 472–477 (1989).

    Article  CAS  Google Scholar 

  28. Laio, A., VandeVondele, J. & Rothlisberger, U. A Hamiltonian electrostatic coupling scheme for hybrid Car–Parrinello molecular dynamics simulations. J. Chem. Phys. 116, 6941–6947 (2002).

    Article  CAS  Google Scholar 

  29. Becke, A. D. Density-functional exchange-energy approximation with correct asymptotic behavior. Phys. Rev. A 38, 3098–3100 (1988).

    Article  CAS  Google Scholar 

  30. Lee, C., Yang, W. & Parr, R. G. Development of the Colle–Salvetti correlation-energy formula into a functional of the electron density. Phys. Rev. B 37, 785–789 (1988).

    Article  CAS  Google Scholar 

  31. Dürr, S. L. et al. The role of conserved residues in the DEDDh motif: the proton-transfer mechanism of HIV-1 RNase H. ACS Catal. 11, 7915–7927 (2021).

    Article  Google Scholar 

  32. Casalino, L., Palermo, G., Rothlisberger, U. & Magistrato, A. Who activates the nucleophile in ribozyme catalysis? An answer from the splicing mechanism of group II introns. J. Am. Chem. Soc. 138, 10374–10377 (2016).

    Article  CAS  PubMed  Google Scholar 

  33. Borišek, J. & Magistrato, A. All-atom simulations decrypt the molecular terms of RNA catalysis in the exon-ligation step of the spliceosome. ACS Catal. 10, 5328–5334 (2020).

    Article  Google Scholar 

  34. Palermo, G. et al. Catalytic metal ions and enzymatic processing of DNA and RNA. Acc. Chem. Res. 48, 220–228 (2015).

    Article  CAS  PubMed  Google Scholar 

  35. Gong, S., Yu, H. H., Johnson, K. A. & Taylor, D. W. DNA unwinding is the primary determinant of CRISPR-Cas9 activity. Cell Rep. 22, 359–371 (2018).

    Article  CAS  PubMed  Google Scholar 

  36. Laio, A. & Parrinello, M. Escaping free-energy minima. Proc. Natl Acad. Sci. USA 99, 12562–12566 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Cisneros, G. A. et al. Reaction mechanism of the ε subunit of E. coli DNA polymerase III: insights into active site metal coordination and catalytically significant residues. J. Am. Chem. Soc. 131, 1550–1556 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Wang, Y. et al. Real-time observation of cas9 postcatalytic domain motions. Proc. Natl Acad. Sci. USA 118, e2010650118 (2021).

    Article  CAS  PubMed  Google Scholar 

  39. Palermo, G. Structure and dynamics of the CRISPR–Cas9 catalytic complex. J. Chem. Inf. Model. 59, 2394–2406 (2019).

    Article  CAS  PubMed  Google Scholar 

  40. Galburt, E. A. & Stoddard, B. L. Catalytic mechanisms of restriction and homing endonucleases. Biochemistry 41, 13851–13860 (2002).

    Article  CAS  PubMed  Google Scholar 

  41. Perez, A. et al. Refinement of the AMBER force field for nucleic acids: improving the description of α/γ conformers. Biophys. J. 92, 3817–3829 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Banas, P. et al. Performance of molecular mechanics force fields for RNA simulations: stability of UUCG and GNRA hairpins. J. Chem. Theor. Comput. 6, 3836–3849 (2010).

    Article  CAS  Google Scholar 

  43. Zgarbova, M. et al. Refinement of the Cornell et al. nucleic acids force field based on reference quantum chemical calculations of glycosidic torsion profiles. J. Chem. Theory Comput. 7, 2886–2902 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Li, P., Roberts, B. P., Chakravorty, D. K. & Merz, K. M. Rational design of particle mesh Ewald compatible Lennard-Jones parameters for +2 metal cations in explicit solvent. J. Chem. Theory Comput. 9, 2733–2748 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Berendsen, H. J. C., Postma, J. P. M., van Gunsteren, W. F., DiNola, A. & Haak, J. R. Molecular dynamics with coupling to an external bath. J. Chem. Phys. 81, 3684–3690 (1984).

    CAS  Google Scholar 

  46. Case, D. A. et al. AMBER 2020 (Univ. of California, San Francisco, 2020).

  47. Parrinello, M., Andreoni, W. & Curioni, A. CPMD (IBM Corporation and Max-Planck Institute, 2000).

  48. Hoover, W. G. Canonical dynamics: equilibrium phase-space distributions. Phys. Rev. A 31, 1695–1697 (1985).

    Article  CAS  Google Scholar 

  49. Nosé, S. An extension of the canonical ensemble molecular dynamics method. Mol. Phys. 57, 187–191 (1986).

    Article  Google Scholar 

  50. Car, R. & Parrinello, M. Unified approach for molecular dynamics and density-functional theory. Phys. Rev. Lett. 55, 2471–2474 (1985).

    Article  CAS  PubMed  Google Scholar 

Download references


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.).

Author information

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.

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 peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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 this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

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).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing