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Peer-reviewedCC BY-NC-ND 4.0JACS Au

Visualizing the Atomic-Level Impact of Anti-CRISPR II–C1 Inhibition on Geo Cas9 with Integrated Structural Biophysics

  • Alexa L. Knight1
  • Helen B. Belato1
  • Praise Lasekan1
  • Charlotte S. Dresser1
  • Chinmai Pindi2
  • Briana J. Mercado1
  • Jinping Luo1
  • Gerwald Jogl1
  • Giulia Palermo2
  • George P. Lisi1
  1. 1Brown University , , , ,
  2. 2University of California Los Angeles , , , ,
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Abstract

Anti-CRISPRs (Acrs) are small protein inhibitors of CRISPR-Cas effectors that originate from the translated genetic material of bacteriophage. Harnessing the natural ability of Acrs to bind and disrupt CRISPR-Cas editing can provide enhanced spatiotemporal control of gene editing. Recent studies have revealed diverse structures and functions of Acrs, however, atomistic insight into the specific molecular mechanisms behind Acr inhibition is lacking. Here, we reveal how structure, function, and dynamics govern AcrIIC1 inhibition of Cas9 fromGeobacillus stearothermophilus(GeoCas9) via its HNH nuclease domain. An X-ray crystal structure of the GeoHNH-AcrIIC1complex reveals a conserved binding interface at the catalytic site and disruption of crucial electrostatic contacts known to modulate the thermostability of GeoCas9. AcrIIC1 binding also rewires the intrinsic dynamics of the GeoHNH domain, stimulates millisecond motions that are absent from the unliganded nuclease, and attenuates the guide RNA affinity of GeoCas9. Subsequent AcrIIC1 mutations in residues at its crystallographic binding interface uncouple Acr binding from inhibition, providing new insight into mechanism by which AcrIIC1 acts on GeoCas9.

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Cite

BibTeXRIS
BibTeX
@article{knight2026visualizing,
  title = {{Visualizing the Atomic-Level Impact of Anti-CRISPR II–C1 Inhibition on Geo Cas9 with Integrated Structural Biophysics}},
  author = {Alexa L. Knight and Helen B. Belato and Praise Lasekan and Charlotte S. Dresser and Chinmai Pindi and Briana J. Mercado and Jinping Luo and Gerwald Jogl and Giulia Palermo and George P. Lisi},
  journal = {JACS Au},
  year = {2026},
  month = oct,
  doi = {10.1021/jacsau.6c00870},
  url = {https://pubs.acs.org/jaaucr/article/doi/10.1021/jacsau.6c00870/5456185/Visualizing-the-Atomic-Level-Impact-of-Anti-CRISPR},
  note = {CC BY-NC-ND 4.0},
}
RIS
TY  - JOUR
TI  - Visualizing the Atomic-Level Impact of Anti-CRISPR II–C1 Inhibition on Geo Cas9 with Integrated Structural Biophysics
AU  - Alexa L. Knight
AU  - Helen B. Belato
AU  - Praise Lasekan
AU  - Charlotte S. Dresser
AU  - Chinmai Pindi
AU  - Briana J. Mercado
AU  - Jinping Luo
AU  - Gerwald Jogl
AU  - Giulia Palermo
AU  - George P. Lisi
PY  - 2026
DA  - 2026/10/22
JO  - JACS Au
DO  - 10.1021/jacsau.6c00870
UR  - https://pubs.acs.org/jaaucr/article/doi/10.1021/jacsau.6c00870/5456185/Visualizing-the-Atomic-Level-Impact-of-Anti-CRISPR
AB  - Anti-CRISPRs (Acrs) are small protein inhibitors of CRISPR-Cas effectors that originate from the translated genetic material of bacteriophage. Harnessing the natural ability of Acrs to bind and disrupt CRISPR-Cas editing can provide enhanced spatiotemporal control of gene editing. Recent studies have revealed diverse structures and functions of Acrs, however, atomistic insight into the specific molecular mechanisms behind Acr inhibition is lacking. Here, we reveal how structure, function, and dynamics govern AcrIIC1 inhibition of Cas9 fromGeobacillus stearothermophilus(GeoCas9) via its HNH nuclease domain. An X-ray crystal structure of the GeoHNH-AcrIIC1complex reveals a conserved binding interface at the catalytic site and disruption of crucial electrostatic contacts known to modulate the thermostability of GeoCas9. AcrIIC1 binding also rewires the intrinsic dynamics of the GeoHNH domain, stimulates millisecond motions that are absent from the unliganded nuclease, and attenuates the guide RNA affinity of GeoCas9. Subsequent AcrIIC1 mutations in residues at its crystallographic binding interface uncouple Acr binding from inhibition, providing new insight into mechanism by which AcrIIC1 acts on GeoCas9.
SN  - 2691-3704
ER  -