1. Conformational control of Cas9 by CRISPR hybrid RNA-DNA guides mitigates off-target activity in T cells
- Author
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David B. Nyer, Lynda M. Banh, Megan van Overbeek, Tomer Rotstein, Elaine Lau, Kevin Baek, Chris R. Fuller, Martin Pacesa, Euan M. Slorach, Andrew May, Martin Jinek, Mckenzi S. Toh, B. Kohrs, Jason Gibson, Bastien Vidal, Matthew J. Irby, Arthur L.G. Owen, Paul Daniel Donohoue, and Peter Cameron
- Subjects
T-Lymphocytes ,CRISPR-Associated Proteins ,Allosteric regulation ,Molecular Conformation ,Computational biology ,Cleavage (embryo) ,Structure-Activity Relationship ,03 medical and health sciences ,0302 clinical medicine ,Genome editing ,CRISPR-Associated Protein 9 ,Humans ,CRISPR ,Molecular Biology ,030304 developmental biology ,Gene Editing ,0303 health sciences ,Nuclease ,Genome ,biology ,Cas9 ,DNA–DNA hybridization ,DNA ,Genomics ,Cell Biology ,Endonucleases ,Genetic Techniques ,Duplex (building) ,Leukocytes, Mononuclear ,biology.protein ,CRISPR-Cas Systems ,030217 neurology & neurosurgery ,RNA, Guide, Kinetoplastida - Abstract
Summary 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.
- Published
- 2021
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