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

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. [Display omitted] •chRDNAs improve Cas9 specificity while preserving on-target editing efficiency•2′-Deoxynucleotide positioning affects guide specificity in a target-dependent manner•chRDNAs cause distorted guide-target DNA duplex geometry and R-loop destabilization•chRDNAs slow Cas9 cleavage rates and promote dissociation of off-target substrates Cas9 off-target activity remains a concern for therapeutics. Donohoue, Pacesa, et al. demonstrate that selective 2′-deoxynucleotide modifications of the Cas9 guide reduce off-target activity in primary T cells. These CRISPR hybrid RNA-DNAs (chRDNAs) increase specificity by distorting the guide-substrate duplex, which disfavors the binding and cleavage of off-target substrates.