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

• Main Authors: Zhicheng Zuo, Ashwini Zolekar, Kesavan Babu, Victor JT Lin, Hamed S Hayatshahi, Rakhi Rajan, Yu-Chieh Wang, Jin Liu
• Format: Article
• Language: English
• Published: eLife Sciences Publications Ltd 2019-07-01
• Series: eLife
• Subjects: HEK293T cell; CRISPR-Cas9; catalysis model
• Online Access: https://elifesciences.org/articles/46500

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.