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, is still controversial. Here, quantum–classical molecular dynamics 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 an associative pathway fundamentally assisted by the joint dynamics of the two Mg2+ ions. This reconciles previous controversial experimental evidences, which could not fully establish the catalytic role of the conserved H983 and the metal cluster conformation. On the other hand, free energy simulations of the crystallographic configuration reveal a phosphate-mediated catalysis that is unlikely to achieve DNA cleavages. Overall, the catalysis of DNA cleavage described here resolves a fundamental open question in the CRISPR-Cas9 biology, which has remained unclear from previous experimental evidences. These findings are valuable for improving the catalytic efficiency and the specificity of CRISPR-Cas9, which is at the basis of the development of novel genome-editing tools.

Ab-initio molecular dynamics reveals the catalysis of DNA cleavage in CRISPR-Cas9

ACS Fall 2020 Virtual Meeting

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