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

ELife
Zhicheng ZuoJin Liu

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.

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Citations

Oct 28, 2020·Journal of Chemical Information and Modeling·Aakash SahaGiulia Palermo
Aug 21, 2021·Annual Review of Genetics·Chunyu Liao, Chase L Beisel

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Datasets Mentioned

BETA
UA159
SKU08691002

Methods Mentioned

BETA
flow cytometry
electrophoresis
X-ray
in vitro transcription

Software Mentioned

AMBER16
AMBER18
AMBER16 pmemd engine
Cluster Omega
AMBERTOOL16
MMPBSA
ImageJ
MSAViewer
GBSA
DFTB3

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