CRISPR-Cas9 is a “molecular scissor” bearing innovative discoveries across the field of Life Sciences. At the molecular level, Cas9 is a metal-dependent enzyme that shows an intriguing biochemical activity. Mg 2+ , Mn 2+ and Ca 2+ have been experimentally shown to not only affect the catalytic activity of the enzyme, but exert a remarkable effect on the protein conformational activation. Here, we present an in-depth study that combines multi-μs classical molecular dynamics (MD) simulations and enhanced sampling methods to gain information into the biophysical role of multiple metal ions. We reveal that an excess in metal ions results in critical interactions fostering the approach of the nuclease lobe to the cleavage site. Regardless the nature of the metal ion, how the ionic strength influences conformational changes in Cas9, in agreement with experimental studies based on biochemical and single-molecule experiments. Mixed quantum/classical methods and ab-initio MD have also been employed, in combination with metadynamics to characterize the catalytic mechanism. Taken together, our study improves the understanding of the metal dependency of Cas9, from its biophysical function to the chemical mechanism. These outcomes can help the design of novel Cas9-based genome editing systems for application in cells and organisms that exhibit diverse ions uptake, metabolism and concentration.

Defining the metal dependent biophysics of CRISPR-Cas9 though multiscale models

ACS Spring 2022

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