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

Zhicheng Zuo,Victor Jt Lin,Kesavan Babu,Rakhi Rajan,Ashwini Zolekar,Hamed S Hayatshahi,Yu-Chieh Wang,Jin Liu

doi:10.7554/elife.46500

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.

Highlights

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 from Streptococcus pyogenes (SpyCas9) has become a gene-editing tool that holds an immense promise for the development of novel therapeutic approaches for human diseases (Cong et al, 2013; Jinek et al, 2012; Knott and Doudna, 2018; Mali et al, 2013)
Intrigued by the above paradoxical findings between the structural and functional experiments, we performed molecular modeling and molecular dynamics (MD) simulations to further investigate the residues that may participate in the catalysis of target DNA strand (tDNA) cleavage
One noticeable feature in this apo-Cas9 structure is that the a-helical element in the HNH domain bba-metal fold appears to pose a unique conformation with N863 pointing toward the catalytic center (Figure 1—figure supplement 4a)

Summary

Introduction

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas from Streptococcus pyogenes (SpyCas9) has become a gene-editing tool that holds an immense promise for the development of novel therapeutic approaches for human diseases (Cong et al, 2013; Jinek et al, 2012; Knott and Doudna, 2018; Mali et al, 2013). The findings were used to generate a computational model of Cas and this model predicted that the HNH domain relies on a group of three amino acids known collectively as D839-H840-N863 to cleave DNA strands This knowledge is useful to understand exactly how Cas modifies genetic information. We mutated D861 to alanine and tested the activity of the D861A variant using an experimental approach based on Cas9-mediated disruption of the egfp gene in EGFP-expressing human cells This Cas variant exhibited DNA-cleavage activity level similar to that of the wild-type protein (Figure 1e and Figure 1—figure supplement 1). (e) The expression and DNA-editing activity of the wild-type and D861A variants of Cas paired with an sgRNA sequence that targets the egfp gene in HEK293T-EGFP cells.

Results and discussion

Conclusion

Materials and methods