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

Zhicheng Zuo, Ashwini Zolekar, Kesavan Babu, Victor J.T. Lin, Hamed S. Hayatshahi, Rakhi Rajan, Yu Chieh Wang, Jin Liu

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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, D839-H840-N854, 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.

Original languageEnglish
Article numbere46500
Publication statusPublished - Jul 2019


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