CRISPR−Cas9 is a bacterial immune system with exciting applications for genome editing. In spite of extensive experimental characterization, the active site chemistry of the RuvC domain, which performs DNA cleavages, has remained elusive. Here, we deliver an in-depth characterization by using Car-Parrinello Molecular Dynamics (MD) simulations and a mixed Quantum Mechanics/Molecular Mechanics (QM/MM) approach, in combination with a Gaussian accelerated MD method and bioinformatics analysis. We disclose a metal-aided architecture in the RuvC active site, which is poised to operate DNA cleavages, in analogy with other DNA/RNA processing enzymes. Remarkably, the formation of a catalytically competent state of the RuvC domain is only observed upon the full conformational activation of the protein, such allowing concerted cleavages of double stranded DNA. This structure is in agreement with the available experimental data and remarkably differs from previous models based on classical mechanics, demonstrating also that only quantum mechanical simulations can accurately describe the metal-aided active site in CRISPR−Cas9. This fully catalytic structure is to perform DNA cleavage and poses the basis for novel experimental verifications and for engineering efforts aimed at improving the genome editing capability of CRISPR−Cas9.

Catalytic structure and function of CRISPR-Cas9 revealed by <i>ab intio </i>quantum mechanics/molecular mechanics (QM/MM) simulations

2019 Fall National Meeting

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Catalytic structure and function of CRISPR-Cas9 revealed by ab intio quantum mechanics/molecular mechanics (QM/MM) simulations