Principles of target DNA cleavage and the role of Mg2+ in the catalysis of CRISPR–Cas9

Abstract

At the core of the CRISPR-Cas9 genome-editing technology, the endonuclease Cas9 introduces sitespecific breaks in DNA. However, precise mechanistic information to ameliorating Cas9 function is still missing. Here, multi-microsecond molecular dynamics, free-energy and multiscale simulations are combined with solution NMR and DNA cleavage experiments to resolve the catalytic mechanism of target DNA cleavage. We show that the conformation of an active HNH nuclease is tightly dependent on the catalytic Mg 2+ , unveiling its cardinal structural role. This activated Mg 2+ -bound HNH is consistently described through molecular simulations, solution NMR and DNA cleavage assays, revealing also that the protonation state of the catalytic H840 is strongly affected by active site mutations. Finally, ab-initio QM(DFT)/MM simulations and metadynamics establish the catalytic mechanism, showing that the catalysis is activated by H840 and completed by K866, rationalising DNA cleavage experiments. This information is critical to enhance the enzymatic function of CRISPR-Cas9 toward improved genome-editing.

Twisting and swiveling domain motions in Cas9 to recognize target DNA duplexes, make double-strand breaks, and release cleaved duplexes

“…frontiersin.org water by H840, followed by catalysis through a concerted associative mechanism similar to RuvC (Nierzwicki et al, 2022). Interestingly, the water molecule coordinating the Mg 2+ ion was seen to shuttle a proton from K866 to the DNA O3' to form the final product, suggesting a possible catalytic role of K866.…”

“…An alternative catalytic mechanism was also proposed where N863 does not engage in the metal coordination and the catalytic water comes from the second shell of metal ion coordination ( Zhao et al, 2020 ). Ab initio QM/MM studies of the tDNA cleavage revealed the activation of nucleophilic water by H840, followed by catalysis through a concerted associative mechanism similar to RuvC ( Nierzwicki et al, 2022 ). Interestingly, the water molecule coordinating the Mg 2+ ion was seen to shuttle a proton from K866 to the DNA O3’ to form the final product, suggesting a possible catalytic role of K866.…”

“…Interestingly, the water molecule coordinating the Mg 2+ ion was seen to shuttle a proton from K866 to the DNA O3’ to form the final product, suggesting a possible catalytic role of K866. Despite having little effect on the p K a of the catalytic H840 when measured by NMR, mutation of K866 to alanine (K866A) showed a remarkable reduction in enzymatic activity ( Nierzwicki et al, 2022 ). Thus, the quantum mechanics level studies, combined with NMR and biochemical studies, have assisted in the identification of the critical second-shell residues in metal-dependent enzymes.…”

“…Experimentally, it is relatively easy to visualize the relative stable enzyme-substrate or enzyme-product complex during the action of catalysis but is nearly impossible to do so for any enzyme/non-cognate substrate complex during the action of being rejected. In this sense, computational biophysics in conjunction with biochemical and biophysical experiments could provide valuable mechanistic insights into substrate specificity and allostery at the detailed molecular level as discussed here ( Wodak et al, 2019 ; East et al, 2020b ; Nierzwicki et al, 2021 ; Belato et al, 2022 ; Nierzwicki et al, 2022 ).…”

An Alpha-helical Lid Guides the Target DNA toward Catalysis in CRISPR-Cas12a

“…39 The Li & Merz model also reported a good description of Mg 2+ bound sites, in agreement with quantum/classical simulations. 28,40 An integration time step of 2 fs was employed. All bond lengths involving hydrogen atoms were constrained using the SHAKE algorithm.…”