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.
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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 reasonable request. Source data are provided with this paper.
References
Doudna, J. A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096 (2014).
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Jinek, M. et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 343, 1247997 (2014).
Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014).
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).
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).
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).
Sternberg, S. H., LaFrance, B., Kaplan, M. & Doudna, J. A. Conformational control of DNA target cleavage by CRISPR–Cas9. Nature 527, 110–113 (2015).
Biertümpfel, C., Yang, W. & Suck, D. Crystal structure of T4 endonuclease VII resolving a Holliday junction. Nature 449, 616–620 (2007).
Zuo, Z. & Liu, J. Structure and dynamics of Cas9 HNH domain catalytic state. Sci. Rep. 7, 17271 (2017).
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).
Jiang, F. et al. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage. Science 351, 867–871 (2016).
Huai, G. et al. Structural insights into DNA cleavage activation of CRISPR-Cas9 system. Nat. Commun. 8, 1375 (2017).
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).
Bravo, J. P. K. et al. Structural basis for mismatch surveillance by CRISPR–Cas9. Nature 603, 343–347 (2022).
Pacesa, M. et al. R-loop formation and conformational activation mechanisms of Cas9. Nature 609, 191–96 (2022).
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).
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).
Zhao, L. N., Mondal, D. & Warshel, A. Exploring alternative catalytic mechanisms of the Cas9 HNH domain. Proteins 88, 260–264 (2019).
Kästner, J. Umbrella sampling. WIREs Comput. Mol. Sci. 1, 932–942 (2011).
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).
Nierzwicki, Ł. et al. Enhanced specificity mutations perturb allosteric signaling in the CRISPR-Cas9 HNH endonuclease. eLife 10, e73601 (2021).
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).
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).
Shimahara, H. et al. Tautomerism of histidine 64 associated with proton transfer in catalysis of carbonic anhydrase. J. Biol. Chem. 282, 9646–9656 (2007).
Brunk, E. et al. Pushing frontiers of first-principles based computer simulations of chemical and biological systems. Chimia (Aarau) 65, 667–671 (2011).
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).
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).
Becke, A. D. Density-functional exchange-energy approximation with correct asymptotic behavior. Phys. Rev. A 38, 3098–3100 (1988).
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).
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).
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).
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).
Palermo, G. et al. Catalytic metal ions and enzymatic processing of DNA and RNA. Acc. Chem. Res. 48, 220–228 (2015).
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).
Laio, A. & Parrinello, M. Escaping free-energy minima. Proc. Natl Acad. Sci. USA 99, 12562–12566 (2002).
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).
Wang, Y. et al. Real-time observation of cas9 postcatalytic domain motions. Proc. Natl Acad. Sci. USA 118, e2010650118 (2021).
Palermo, G. Structure and dynamics of the CRISPR–Cas9 catalytic complex. J. Chem. Inf. Model. 59, 2394–2406 (2019).
Galburt, E. A. & Stoddard, B. L. Catalytic mechanisms of restriction and homing endonucleases. Biochemistry 41, 13851–13860 (2002).
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).
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).
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).
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).
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).
Case, D. A. et al. AMBER 2020 (Univ. of California, San Francisco, 2020).
Parrinello, M., Andreoni, W. & Curioni, A. CPMD (IBM Corporation and Max-Planck Institute, 2000).
Hoover, W. G. Canonical dynamics: equilibrium phase-space distributions. Phys. Rev. A 31, 1695–1697 (1985).
Nosé, S. An extension of the canonical ensemble molecular dynamics method. Mol. Phys. 57, 187–191 (1986).
Car, R. & Parrinello, M. Unified approach for molecular dynamics and density-functional theory. Phys. Rev. Lett. 55, 2471–2474 (1985).
Acknowledgements
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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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.
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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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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). https://doi.org/10.1038/s41929-022-00848-6
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DOI: https://doi.org/10.1038/s41929-022-00848-6
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