Related Experiment Video
Updated: Jun 9, 2025
08:23
CIRCLE-Seq for Interrogation of Off-Target Gene Editing
Published on: November 1, 2024
534
Dynamic sampling of a surveillance state enables DNA proofreading by Cas9
Viviane S De Paula1, Abhinav Dubey2, Haribabu Arthanari2
1Center for Computational and Genomic Medicine, The Children's Hospital of Philadelphia, 3501 Civic Center Boulevard, Philadelphia, PA 19104, USA; Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, 3620 Hamilton Walk, Philadelphia, PA 19104-6059, USA.
Cell Chemical Biology
|October 29, 2024
Summary
CRISPR-Cas9
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- CRISPR-Cas9 technology enables genome engineering via guide RNA programming.
- The exact mechanism of DNA target discrimination by Cas9 is not fully understood.
Purpose of the Study:
- To elucidate the molecular mechanism of DNA mismatch recognition by CRISPR-Cas9.
- To investigate the role of protein conformation in Cas9 specificity.
Main Methods:
- Methyl-based Nuclear Magnetic Resonance (NMR) spectroscopy was used.
- Holoenzymes were assembled in vitro to study CRISPR-Cas9-DNA interactions.
Main Results:
- A specific protein conformational state was identified that recognizes DNA mismatches.
- An allosteric pathway links the REC3 domain's conformational switch to the HNH domain's catalytic state.
- HiFi Cas9 (R691A) enhances DNA recognition fidelity by stabilizing a 'surveillance state'.
Conclusions:
- CRISPR-Cas9 selectivity is governed by an allosteric protein-based switch.
- Understanding this mechanism provides insights into Cas9's molecular function and specificity.
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