Key role of the REC lobe during CRISPR–Cas9 activation by ‘sensing’, ‘regulating’, and ‘locking’ the catalytic HNH domain
Open accessAbstract
Understanding the conformational dynamics of CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 is of the utmost importance for improving its genome editing capability. Here, molecular dynamics simulations performed using Anton-2 - a specialized supercomputer capturing micro-to-millisecond biophysical events in real time and at atomic-level resolution - reveal the activation process of the endonuclease Cas9 toward DNA cleavage. Over the unbiased simulation, we observe that the spontaneous approach of the catalytic domain HNH to the DNA cleavage site is accompanied by a remarkable structural remodeling of the recognition (REC) lobe, which exerts a key role for DNA cleavage. Specifically, the significant conformational changes and the collective conformational dynamics of the REC lobe indicate a mechanism by which the REC1-3 regions 'sense' nucleic acids, 'regulate' the HNH conformational transition, and ultimately 'lock' the HNH domain at the cleavage site, contributing to its catalytic competence. By integrating additional independent simulations and existing experimental data, we provide a solid validation of the activated HNH conformation, which had been so far poorly characterized, and we deliver a comprehensive understanding of the role of REC1-3 in the activation process. Considering the importance of the REC lobe in the specificity of Cas9, this study poses the basis for fully understanding how the REC components control the cleavage of off-target sequences, laying the foundation for future engineering efforts toward improved genome editing.
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PAM binding to CRISPR-Cas9 plays a key role in triggering the interdependent conformational dynamics of HNH and RuvC, likely enabling the concerted cleavage of the DNA strands.
“This led the authors to conclude that PAM binding to CRISPR-Cas9 plays a key role in triggering the interdependent conformational dynamics of HNH and RuvC, likely enabling the concerted cleavage of the DNA strands (Palermo et al., 2018).”
From Allostery in Its Many Disguises: From Theory to Applications (2019) · cites this paper as Palermo et al., 2018
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- Palermo G, Chen JS, Ricci CG, Rivalta I, Jinek M, Batista VS, Doudna JA, McCammon JA (2018). Key role of the REC lobe during CRISPR–Cas9 activation by ‘sensing’, ‘regulating’, and ‘locking’ the catalytic HNH domain. Quarterly Reviews of Biophysics 51, e9, 1– 11. https://doi.org/10.1017/S0033583518000070 PMC6292676 30555184
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- Authors
- Palermo, G., Chen, J. S., Ricci, C. G., Rivalta, I., Jinek, M., Batista, V. S., Doudna, J. A., & McCammon, J. A.
- Affiliations
- University of California, Riverside (US)
- University of California, Berkeley (US)
- University of California San Diego (US)
- Lyon 1 Université (FR)
- Howard Hughes Medical Institute (US)
- San Diego Supercomputer Center (US)
- Corresponding author
- Palermo, Giulia
- ORCID
- Venue
- Quarterly Reviews of Biophysics
- Publisher
- Cambridge University Press (CUP)
- Published
- 2018
- Volume
- 51
- ISSN
- 0033-5835 (ISSN-L)
- 1469-8994
- PMC
- PMC6292676
- OpenAlex
- W2885445984
- Cited by
- 123
- References
- 61
- Citations by year
- 2026: 8 · 2025: 25 · 2024: 16 · 2023: 11 · 2022: 21 · 2021: 11 · 2020: 11 · 2019: 17 · 2018: 3
- FWCI
- 1.03
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Locations
- Quarterly Reviews of Biophysics (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Zurich Open Repository and Archive (University of Zurich) (acceptedVersion, cc-by)
- Europe PMC (PubMed Central) (acceptedVersion, cc-by)
- PubMed Central (acceptedVersion, cc-by)
- eScholarship (California Digital Library) (submittedVersion)
- OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
- HAL (submittedVersion)
- Archivio istituzionale della ricerca (Alma Mater Studiorum Università di Bologna) (submittedVersion, cc-by)
- Zurich Open Repository and Archive (University of Zurich) (submittedVersion, cc-by)
- Zurich Open Repository and Archive (University of Zurich)
- Funders
- National Science Foundation (MCB160059)
- Howard Hughes Medical Institute
- San Diego Supercomputer Center (MCB160059)
- Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (164698)
- National Institutes of Health
- National Institute of General Medical Sciences (R01GM031749)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Keywords
- CRISPR/Cas9, DNA cleavage, endonuclease activation, conformational dynamics, off-target specificity, genome editing, molecular dynamics simulation
- MeSH terms
- Gene Editing, CRISPR-Associated Protein 9, Humans, Catalytic Domain, Principal Component Analysis, DNA Cleavage, Molecular Dynamics Simulation, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems
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