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

Lorenzo Casalino; Łukasz Nierzwicki; Martin Jinek; Giulia Palermo

doi:10.1021/acscatal.0c03566

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

Abstrakt

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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Autorzy (4)

Lorenzo Casalino dr
Łukasz Nierzwicki dr inż.
Martin Jinek dr
Giulia Palermo dr

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Wersja publikacji: Accepted albo Published Version
Licencja: Copyright (2020 American Chemical Society)

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Informacje szczegółowe

Kategoria:: Publikacja w czasopiśmie
Typ:: artykuły w czasopismach
Opublikowano w:: ACS Catalysis nr 10, strony 13596 - 13605,
ISSN: 2155-5435
Język:: angielski
Rok wydania:: 2020
Opis bibliograficzny:: 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:: Cyfrowy identyfikator dokumentu elektronicznego (otwiera się w nowej karcie) 10.1021/acscatal.0c03566
Weryfikacja:: Politechnika Gdańska