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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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Authors (4)
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Lorenzo Casalino dr
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Martin Jinek dr
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Giulia Palermo dr
Cite as
Full text
- Publication version
- Accepted or Published Version
- License
- Copyright (2020 American Chemical Society)
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Details
- Category:
- Articles
- Type:
- artykuły w czasopismach
- Published in:
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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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