CRISPR-based genome editing of primary human T cells has the potential to revolutionize disease-modifying therapies. However, substantial improvements in CRISPR-Cas9 specificity are needed to minimize its off-target activity in cells. A number of “high-fidelity” Cas9 variants have been engineered in an attempt to address specificity improvements, but these proteins often are constrained by efficiency-specificity trade-offs, limiting their therapeutic application. Here we show that CRISPR hybrid RNA-DNA (chRDNA) guides composed of both RNA and DNA nucleotides are a highly effective approach to increase Cas9 specificity while preserving on-target editing activity. Across multiple genomic targets in therapeutically relevant primary human T cells, we show that 2’-deoxynucleotide (dnt) positioning affects guide activity in a sequence-dependent manner, and that this can be leveraged to engineer chRDNA guides with minimal to no detectable off-target activities. To gain mechanistic insight into chRDNA activity, we performed structural analysis of Cas9-chRDNA complexes, revealing that chRDNA guides adopt distorted helical conformations upon target hybridization to disfavor engagement of off-target sequences. Strikingly, through iterative engineering of dnt number and position in the chRDNA design, we fine-tuned specificity to discriminate between sequences that differ by only a single nucleotide, which was not achieved using “high-fidelity” Cas9 variants. Together, these results demonstrate that chRDNAs are designed for highly efficient and precise T cell genome editing, paving the way for their application in therapeutics.

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

ACS Spring 2022

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