Structural basis for Cas9 off-target activity
Open accessPublished version of a preprint
Abstract
The target DNA specificity of the CRISPR-associated genome editor nuclease Cas9 is determined by complementarity to a 20-nucleotide segment in its guide RNA. However, Cas9 can bind and cleave partially complementary off-target sequences, which raises safety concerns for its use in clinical applications. Here, we report crystallographic structures of Cas9 bound to bona fide off-target substrates, revealing that off-target binding is enabled by a range of noncanonical base-pairing interactions within the guide:off-target heteroduplex. Off-target substrates containing single-nucleotide deletions relative to the guide RNA are accommodated by base skipping or multiple noncanonical base pairs rather than RNA bulge formation. Finally, PAM-distal mismatches result in duplex unpairing and induce a conformational change in the Cas9 REC lobe that perturbs its conformational activation. Together, these insights provide a structural rationale for the off-target activity of Cas9 and contribute to the improved rational design of guide RNAs and off-target prediction algorithms.
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Off-target effects remain a major challenge for CRISPR/Cas systems in translational medicine.
“Although CRISPR/Cas systems exhibit tremendous potential in translational medicine, off-target effects remain a major challenge (Fu et al., 2013; Hsu et al., 2013; Pacesa et al., 2022).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Pacesa et al., 2022
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References 97 · Cited by 198
Research works printed but not linked: 1
- Pacesa M, and Jinek M. (2021). Mechanism of R-loop formation and conformational activation of Cas9. bioRxiv, 2021.2009.2016.460614.
- Authors
- Pacesa, M., Lin, C.-H., Cléry, A., Saha, A., Arantes, P. R., Bargsten, K., Irby, M. J., Allain, F. H.-T., Palermo, G., Cameron, P., Donohoue, P. D., & Jinek, M.
- Affiliations
- Caribou Biosciences (United States) (US)
- ETH Zurich (CH)
- University of California, Riverside (US)
- University of Zurich (CH)
- ORCID
- Venue
- Cell
- Publisher
- Elsevier BV
- Published
- 2022-10
- Volume
- 185
- Issue
- 22
- Pages
- 4067-4081.e21
- ISSN
- 0092-8674 (ISSN-L)
- PMC
- PMC10103147
- OpenAlex
- W4307725211
- Cited by
- 198
- References
- 97
- Citations by year
- 2026: 39 · 2025: 69 · 2024: 49 · 2023: 35 · 2022: 6
- FWCI
- 13.06
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Cell (publishedVersion, cc-by-nc-nd)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Zurich Open Repository and Archive (University of Zurich) (acceptedVersion, cc-by-nc-nd)
- DORA PSI (Paul Scherrer Institute) (submittedVersion, cc-by-nc-nd)
- Zurich Open Repository and Archive (University of Zurich)
- Funders
- National Science Foundation (R01GM141329, 1905374)
- Howard Hughes Medical Institute
- Vallee Foundation
- Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (182567)
- Paul Scherrer Institut
- National Institutes of Health
- National Institute of General Medical Sciences (R01GM141329)
- Division of Chemistry (CHE-1905374)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Keywords
- Cas9, off-target effects, CRISPR/Cas9, guide RNA, heteroduplex, nuclease specificity, off-target prediction, guide RNA design, crystal structure
- MeSH terms
- Gene Editing, RNA, Guide, CRISPR-Cas Systems, Endonucleases, Nucleotides, RNA, Guide, Kinetoplastida, Base Pairing, CRISPR-Cas Systems
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