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

Elife. 2019 Jul 30:8:e46500. 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.

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

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • CRISPR-Associated Protein 9 / chemistry*
  • CRISPR-Associated Protein 9 / metabolism*
  • Catalytic Domain
  • DNA / chemistry
  • DNA / metabolism*
  • Molecular Dynamics Simulation
  • Protein Binding
  • Protein Conformation
  • RNA, Guide, CRISPR-Cas Systems / chemistry
  • RNA, Guide, CRISPR-Cas Systems / metabolism*
  • Streptococcus pyogenes / enzymology*

Substances

  • RNA, Guide, CRISPR-Cas Systems
  • DNA
  • CRISPR-Associated Protein 9