Highly Specific Cas9 and Cas12a Engineering of Human T Cells for Generation of Novel Allogeneic Cell Therapies

Introduction 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 significantly reduce its off-target activity in cells. Here we show that CRISPR hybrid RNA-DNA (chRDNA) guides designed with both RNA and DNA nucleotides are a highly effective approach to increase Cas9 specificity while preserving on-target editing activity. Methods and Results Across multiple genomic targets in primary human T cells, we show that 2’-deoxynucleotide (dnt) positioning affects guide activity in a sequence-dependent manner, and we leveraged this observation to engineer chRDNA guides with minimal to no detectable off-target activities (Fig. 1). 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 differentiate between sequences that differ by only a single nucleotide, which was not achieved using “high-fidelity” Cas9 variants. From the combined understanding of Cas9 chRDNA design principles and mechanistic studies, we designed chRDNA guides for use with the Cas12a CRISPR system. Our data showed tailored Cas12a chRDNA guides also demonstrate robust Cas12a-mediated genome editing in primary human T cells without detectable off-target editing (Fig. 2). This observation supports the portability of the chRDNA platform across CRISPR systems and exhibits its advantages in terms of versatility compared to traditional protein engineering approaches. To further enhance the capability of the Cas12a chRDNA platform for engineering of cell therapies, we optimized nuclear trafficking, thereby improving editing efficiency even at low concentrations of Cas12a across multiple primary cell types (Fig. 3). Conclusion These results demonstrate that chRDNAs enable highly efficient and precise genome editing, paving the way for their utilization in clinical development of allogeneic cell therapies. The Cas9 chRDNA genome-editing technology was used to engineer three edits for CB-010, an allogeneic anti-CD19 CAR-T cell therapy being evaluated in the ANTLER trial for relapsed/refractory B cell non-Hodgkin lymphoma patients (NCT04637763), and the Cas12a chRDNA genome-editing technology was used to engineer four edits for CB-011, an allogeneic anti-BCMA CAR-T cell therapy being evaluated in the CaMMouflage trial for relapsed/refractory multiple myeloma patients (NCT05722418).