Molecular cellSep 3, 2021

Using hybrid RNA-DNA guides to control Cas9 shape reduces unwanted DNA cutting in T cells

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Mar 29, 2026

Abstract

CRISPR hybrid RNA-DNA guides can substantially reduce off-target effects of Cas9 nuclease.

High-fidelity Cas9 variants show varying efficiencies and residual off-target effects.
2'-deoxynucleotide positioning in chRDNA guides affects guide activity and specificity based on the target.
Crystal structures of Cas9-chRDNA complexes demonstrate altered guide-target duplex geometry.
These structural changes are linked to increased specificity by affecting DNA binding and Cas9 activation dynamics.
Customized chRDNAs may enhance the safety and effectiveness of therapeutic genome editing.

Simplified

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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Declaration of interests P.D.D., B.V., M.J.I., D.B.N., J.G., A.L.G.O., L.B., B.K., and C.K.F. own shares or stock options and are employees of Caribou Biosciences. E.L., M.S.T., T. R., K.B., A.P.M., E.M.S, M.v.O., and P.C. are shareholders and/or former employees of Caribou Biosciences. M.J. is a co-founder, shareholder, and member of the scientific advisory board of Caribou Biosciences. P.D.D., A.P.M., M.J.I., M.v.O., P.C., and M.J. are named inventors on patents and patent applications related to CRISPR-Cas technologies.

PubMed

Using hybrid RNA-DNA guides to control Cas9 shape reduces unwanted DNA cutting in T cells

Abstract

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Abstract

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Related papers

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