Conformational control of Cas9 by CRISPR hybrid RNA-DNA guides mitigates off-target activity in T cells
Open accessAbstract
The off-target activity of the CRISPR-associated nuclease Cas9 is a potential concern for therapeutic genome editing applications. Although high-fidelity Cas9 variants have been engineered, they exhibit varying efficiencies and have residual off-target effects, limiting their applicability. Here, we show that CRISPR hybrid RNA-DNA (chRDNA) guides provide an effective approach to increase Cas9 specificity while preserving on-target editing activity. Across multiple genomic targets in primary human T cells, we show that 2'-deoxynucleotide (dnt) positioning affects guide activity and specificity in a target-dependent manner and that this can be used to engineer chRDNA guides with substantially reduced off-target effects. Crystal structures of DNA-bound Cas9-chRDNA complexes reveal distorted guide-target duplex geometry and allosteric modulation of Cas9 conformation. These structural effects increase specificity by perturbing DNA hybridization and modulating Cas9 activation kinetics to disfavor binding and cleavage of off-target substrates. Overall, these results pave the way for utilizing customized chRDNAs in clinical applications.
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Hybrid RNA-DNA guides demonstrate significant improvements in gene editing specificity.
“The use of “hybrid” RNA-DNA guides has demonstrated significant improvements in gene editing specificity (Donohoue et al., 2021).”
From Emerging applications of gene editing technologies for the development of climate-resilient crops (2025) · cites this paper as Donohoue et al., 2021
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- Authors
- Donohoue, P. D., Pacesa, M., Lau, E., Vidal, B., Irby, M. J., Nyer, D. B., Rotstein, T., Banh, L., Toh, M. S., Gibson, J., Kohrs, B., Baek, K., Owen, A. L. G., Slorach, E. M., van Overbeek, M., Fuller, C. K., May, A. P., Jinek, M., & Cameron, P.
- Affiliations
- Caribou Biosciences (United States) (US)
- Corresponding author
- Donohoue, Paul D.
- May, Andrew P.
- Cameron, Peter
- ORCID
- Venue
- Molecular Cell
- Publisher
- Elsevier BV
- Published
- 2021-09
- Volume
- 81
- Issue
- 17
- Pages
- 3637-3649.e5
- ISSN
- 1097-2765 (ISSN-L)
- OpenAlex
- W3198847027
- Cited by
- 69
- References
- 60
- Citations by year
- 2026: 11 · 2025: 18 · 2024: 19 · 2023: 8 · 2022: 12 · 2021: 1
- FWCI
- 3.14
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- www.elsevier.com/tdm/userlicense/1.0/ (tdm)
- www.elsevier.com/legal/tdmrep-license (tdm)
- www.elsevier.com/open-access/userlicense/1.0/ (vor, 366 d)
- doi.org/10.15223/policy-017 (stm-asf)
- doi.org/10.15223/policy-037 (stm-asf)
- doi.org/10.15223/policy-012 (stm-asf)
- doi.org/10.15223/policy-029 (stm-asf)
- doi.org/10.15223/policy-004 (stm-asf)
- Locations
- Molecular Cell (publishedVersion)
- PubMed (publishedVersion)
- Zurich Open Repository and Archive (University of Zurich) (acceptedVersion)
- DORA PSI (Paul Scherrer Institute) (submittedVersion)
- Zurich Open Repository and Archive (University of Zurich)
- Funders
- Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (31003A_182567)
- 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
- Health Sciences › Medicine › Oncology › CAR-T cell therapy research
- Keywords
- Cas9, T cells, allosteric modulation, DNA hybridization, genome editing specificity
- MeSH terms
- Gene Editing, CRISPR-Associated Protein 9, RNA, Guide, CRISPR-Cas Systems, DNA, Endonucleases, Genetic Techniques, Humans, Leukocytes, Mononuclear, Molecular Conformation, Structure-Activity Relationship, T-Lymphocytes, Genome, Genomics, CRISPR-Cas Systems, CRISPR-Associated Proteins
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