Allosteric Motions of the CRISPR-Cas9 HNH Nuclease Probed by NMR and Molecular Dynamics

Kyle W. East, Jocelyn C. Newton, Uriel N. Morzan, Yogesh B. Narkhede, Atanu Acharya, Erin Skeens, Gerwald Jogl, Victor S. Batista, Giulia Palermo, George P. Lisi

Research output: Contribution to journalArticlepeer-review

68 Scopus citations


CRISPR-Cas9 is a widely employed genome-editing tool with functionality reliant on the ability of the Cas9 endonuclease to introduce site-specific breaks in double-stranded DNA. In this system, an intriguing allosteric communication has been suggested to control its DNA cleavage activity through flexibility of the catalytic HNH domain. Here, solution NMR experiments and a novel Gaussian-accelerated molecular dynamics (GaMD) simulation method are used to capture the structural and dynamic determinants of allosteric signaling within the HNH domain. We reveal the existence of a millisecond time scale dynamic pathway that spans HNH from the region interfacing the adjacent RuvC nuclease and propagates up to the DNA recognition lobe in full-length CRISPR-Cas9. These findings reveal a potential route of signal transduction within the CRISPR-Cas9 HNH nuclease, advancing our understanding of the allosteric pathway of activation. Further, considering the role of allosteric signaling in the specificity of CRISPR-Cas9, this work poses the mechanistic basis for novel engineering efforts aimed at improving its genome-editing capability.

Original languageEnglish (US)
Pages (from-to)1348-1358
Number of pages11
JournalJournal of the American Chemical Society
Issue number3
StatePublished - Jan 22 2020
Externally publishedYes

ASJC Scopus subject areas

  • Catalysis
  • General Chemistry
  • Biochemistry
  • Colloid and Surface Chemistry


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