Cited by 69CRISPR and Genetic EngineeringRNA regulation and diseaseCAR-T cell therapy research

Conformational control of Cas9 by CRISPR hybrid RNA-DNA guides mitigates off-target activity in T cells

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Donohoue, Paul D., Pacesa, Martin, Lau, Elaine, Vidal, Bastien, Irby, Matthew J., Nyer, David B., Rotstein, Tomer, Banh, Lynda +11 more·2021·Molecular Cell

Abstract

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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Crossref · 10.1016/j.molcel.2021.07.035

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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
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
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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