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Elife. 2019 Jul 30;8. pii: e46500. doi: 10.7554/eLife.46500.

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

Author information

1
Department of Pharmaceutical Sciences, UNT System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, United States.
2
College of Chemistry and Chemical Engineering, Shanghai University of Engineering Science, Shanghai, China.
3
Department of Chemistry and Biochemistry, Price Family Foundation Institute of Structural Biology, Stephenson Life Sciences Research Center, University of Oklahoma, Norman, United States.

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.

KEYWORDS:

CRISPR-Cas9; HEK293T cell; catalysis model; human; molecular biophysics; structural biology

PMID:
31361218
PMCID:
PMC6706240
DOI:
10.7554/eLife.46500
[Indexed for MEDLINE]
Free PMC Article

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