Structural and functional insights into the bona fide catalytic state of Streptococcus pyogenes Cas9 HNH nuclease domain

Abstract

The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (SpyCas9), along with a programmable single-guide RNA (sgRNA), has been exploited as a significant genome-editing tool. Despite the recent advances in determining the SpyCas9 structures and DNA cleavage mechanism, the cleavage-competent conformation of the catalytic HNH nuclease domain of SpyCas9 remains largely elusive and debatable. By integrating computational and experimental approaches, we unveiled and validated the activated Cas9-sgRNA-DNA ternary complex in which the HNH domain is neatly poised for cleaving the target DNA strand. In this catalysis model, the HNH employs the catalytic triad of D839-H840-N863 for cleavage catalysis, rather than previously implicated D839-H840-D861, D837-D839-H840, or D839-H840-D861-N863. Our study contributes critical information to defining the catalytic conformation of the HNH domain and advances the knowledge about the conformational activation underlying Cas9-mediated DNA cleavage.

A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9

“…The pairwise GB model of Hawkins, Cramer, and Truhlar (GB HCT ) was used, adopting the parameters developed by Tsui and Case ( 49 ). Other parameters were set following our previous studies ( 46 ). For the bulge-free systems, the last 50 ns of the replicated trajectories were aggregated for calculations.…”

“…The TGG PAM-containing system was subjected to a sequence of steps to reach stability following our previous protocol (46), involving thorough energy minimization and slow heating to 310.15 K followed by 50-ns equilibration. The equilibrated system was used as the starting point for the TAG and TCGG PAM systems as well as the bulge system with a canonical TGG PAM (see below).…”

Points of View on the Tools for Genome/Gene Editing

“…Either D 10 A [58] or H 983 A [65] mutation destroyed the RuvC nuclease activity. On the other hand, D 839 A [66], H 840 A [58], and N 863 A [67] mutations could eliminate the HNH nuclease activity. These mutations did not influence the target site binding affinity of Cas9-sgRNA.…”

Characterizing the specificity and nicking activity of CRISPR endonucleases, Cas9 and Cas12a

“…Here, DNA unwinding may be the key factor for Cas9 activity (Gong et al, 2018). Structures of Cas9 have also revealed that the HNH domain is mobile Nishimasu et al, 2015Nishimasu et al, , 2014bZhu et al, 2019;Zuo et al, 2019), and the catalytic conformation is preferentially adopted when Cas9 is bound to a target with perfect complementarity al., 2015) ( Figure 2.6D, E). Movement of the HNH domain also allosterically regulates cleavage by the RuvC domain, ensuring high-fidelity cleavage for both strands .…”

“…Cas9 cleavage of the target strand (TS) and non-target strand (NTS) is mediated by the metal ion-dependent HNH and RuvC endonuclease domains, respectively (Gasiunas et al, 2012;Martin Jinek et al, 2012;Zuo et al, 2019) (Figure 2.6E). Recent high-resolution cryo-EM structures of Cas9 reveal three states in the pre-and post-catalysis stages (Zhu et al, 2019).…”