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Casalino_ChemRxiv.pdf (13.27 MB)

Two-Metal Ion Mechanism of DNA Cleavage in CRISPR-Cas9

submitted on 08.09.2019, 08:10 and posted on 10.09.2019, 15:30 by Lorenzo Casalino, Martin Jinek, Giulia Palermo

CRISPR-Cas9 is a cutting-edge genome-editing technology, which employs the endonuclease Cas9 to cleave DNA sequences of interest. However, the catalytic mechanism of DNA cleavage and the critical role of the Mg2+ ions have remained elusive. Here, quantum–classical QM(Car-Parrinello)/MM simulations are used to disclose the two-Mg2+ aided mechanism of phosphodiester bond cleavage in the RuvC domain. We reveal that the catalysis proceeds through an associative pathway activated by H983 and fundamentally assisted by the joint dynamics of the two Mg2+ ions, which cooperatively act to properly orient the reactants and lead the chemical step to completion. Cross-validation of this mechanism is achieved by evaluating alternative reaction pathways and in light of experimental data, delivering fundamental insights on how CRISPR-Cas9 cleaves nucleic acids. This knowledge is critical for improving the Cas9 catalytic efficiency and its metal-dependent function, helping also the development of novel Cas9-based genome-editing tools.


National Science Foundation CHE-1905374



Email Address of Submitting Author


University of California Riverside


United States

ORCID For Submitting Author


Declaration of Conflict of Interest

The authors declare that there is no conflict of interest