Catalytic Mechanism of Non-Target DNA Cleavage in CRISPR-Cas9 Revealed by Ab Initio Molecular Dynamics - Publication - Bridge of Knowledge

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Catalytic Mechanism of Non-Target DNA Cleavage in CRISPR-Cas9 Revealed by Ab Initio Molecular Dynamics

Abstract

CRISPR-Cas9 is a cutting-edge genome editing technology, which uses the endonuclease Cas9 to introduce mutations at desired sites of the genome. This revolutionary tool is promising to treat a myriad of human genetic diseases. Nevertheless, the molecular basis of DNA cleavage, which is a fundamental step for genome editing, has not been established. Here, quantum–classical molecular dynamics (MD) and free energy methods are used to disclose the two-metal-dependent mechanism of phosphodiester bond cleavage in CRISPR-Cas9. Ab initio MD reveals a conformational rearrangement of the Mg2+-bound RuvC active site, which entails the relocation of H983 to act as a general base. Then, the DNA cleavage proceeds through a concerted associative pathway fundamentally assisted by the joint dynamics of the two Mg2+ ions. This clarifies previous controversial experimental evidence, which could not fully establish the catalytic role of the conserved H983 and the metal cluster conformation. The comparison with other two-metal-dependent enzymes supports the identified mechanism and suggests a common catalytic strategy for genome editing and recombination. Overall, the non-target DNA cleavage catalysis described here resolves a fundamental open question in the CRISPR-Cas9 biology and provides valuable insights for improving the catalytic efficiency and the metal-dependent function of the Cas9 enzyme, which are at the basis of the development of genome editing tools.

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Copyright (2020 American Chemical Society)

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Category:
Articles
Type:
artykuły w czasopismach
Published in:
ACS Catalysis no. 10, pages 13596 - 13605,
ISSN: 2155-5435
Language:
English
Publication year:
2020
Bibliographic description:
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 Catalysis -Vol. 10,iss. 22 (2020), s.13596-13605
DOI:
Digital Object Identifier (open in new tab) 10.1021/acscatal.0c03566
Verified by:
Gdańsk University of Technology

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