Active-Site Models of Streptococcus pyogenes Cas9 in DNA Cleavage State

Honghai Tang; Hui Yuan; Wenhao Du; Gan Li; Dongmei Xue; Qiang Huang

doi:10.3389/fmolb.2021.653262

Active-Site Models of Streptococcus pyogenes Cas9 in DNA Cleavage State

Front Mol Biosci. 2021 Apr 21:8:653262. doi: 10.3389/fmolb.2021.653262. eCollection 2021.

Authors

Honghai Tang¹, Hui Yuan¹, Wenhao Du¹, Gan Li¹, Dongmei Xue¹, Qiang Huang^{1

2}

Affiliations

¹ State Key Laboratory of Genetic Engineering, Shanghai Engineering Research Center of Industrial Microorganisms, Ministry of Education Engineering Research Centre of Gene Technology, School of Life Sciences, Fudan University, Shanghai, China.
² Multiscale Research Institute of Complex Systems, Fudan University, Shanghai, China.

Abstract

CRISPR-Cas9 is a powerful tool for target genome editing in living cells. Significant advances have been made to understand how this system cleaves target DNA. HNH is a nuclease domain, which shares structural similarity with the HNH endonuclease characterzied by a beta-beta-alpha-metal fold. Therefore, based on one- and two-metal-ion mechanisms, homology modeling and molecular dynamics (MD) simulation are suitable tools for building an atomic model of Cas9 in the DNA cleavage state. Here, by modeling and MD, we presented an atomic model of SpCas9-sgRNA-DNA complex with the cleavage state. This model shows that the HNH and RuvC conformations resemble their DNA cleavage state where the active-sites in the complex coordinate with DNA, Mg²⁺ ions, and water. Among them, residues D10, E762, H983, and D986 locate at the first shell of the RuvC active-site and interact with the ions directly, residues H982 or/and H985 are general (Lewis) bases, and the coordinated water is located at the positions for nucleophilic attack of the scissile phosphate. Meanwhile, this catalytic model led us to engineer a new SpCas9 variant (SpCas9-H982A + H983D) with reduced off-target effects. Thus, our study provided new mechanistic insights into the CRISPR-Cas9 system in the DNA cleavage state and offered useful guidance for engineering new CRISPR-Cas9 editing systems with improved specificity.

Keywords: CRISPR-Cas9; DNA cleavage mechanism; gene editing; molecular dynamics; off-target effects.