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

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

doi:10.3389/fmolb.2021.653262

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. However, due to difficulty in determining active CRISPR-Cas9 structure in DNA cleavage state by X-ray and cryo-EM, it remains uncertain how the HNH and RuvC nuclease domains in CRISPR-Cas9 split the DNA phosphodiester bonds with metal ions and water molecules. Therefore, based on one-and two-metal-ion mechanisms, homology modeling and molecular dynamics simulation (MD) 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 new SpCas9 variant (SpCas9-H982A + H983D) with reduced off-target effects. Thus, our study provides new mechanistic insights into the CRISPR-Cas9 system in the DNA cleavage state, and offers useful guidance for engineering new CRISPR-Cas9 editing systems with improved specificity.

Highlights

The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely used as a powerful and versatile tool for genome engineering (Hsu et al, 2014; Wang et al, 2016; Chen and Doudna, 2017)
Structural and biochemical studies have suggested that the catalytic residues in the HNH active sites are D839, H840, and N863, which may employ the one-metal-ion mechanism to hydrolyze the complementary strand of the target DNA (Jinek et al, 2012; Nishimasu et al, 2014), in agreement with recent research (Zhu et al, 2019)
Some studies reported that D10, E762, H983, and D986 are the catalytic residues of the RuvC domain, and suggested that they utilize the two-metal-ion hydrolysis mechanism to split the non-complementary strand (De Vivo et al, 2008; Ho et al, 2010; Nishimasu et al, 2014; Palermo et al, 2015; Chen and Doudna, 2017)

Summary

Introduction

The RNA-guided CRISPR-Cas nuclease from Streptococcus pyogenes (SpCas9) has been widely used as a powerful and versatile tool for genome engineering (Hsu et al, 2014; Wang et al, 2016; Chen and Doudna, 2017). Some studies reported that D10, E762, H983, and D986 are the catalytic residues of the RuvC domain, and suggested that they utilize the two-metal-ion hydrolysis mechanism to split the non-complementary (nontarget) strand (De Vivo et al, 2008; Ho et al, 2010; Nishimasu et al, 2014; Palermo et al, 2015; Chen and Doudna, 2017) Unlike those of the HNH residues, the precise roles of these RuvC residues in the DNA cleavage remain debated.

Methods

Results

Conclusion