Orthogonal CRISPR systems for targeted integration and multiplex base editing enable nonviral engineering of...

Multiple genomic modifications, including targeted transgene integrations and knockouts, may be required to develop potent, allogeneic chimeric antigen receptor (CAR)-T cell therapies. Conventional CRISPR-Cas systems generate double-strand breaks (DSBs) associated with genomic rearrangements and genotoxicities. DSB-free base editing reduces these risks. Here, we facilitate multiplex editing by combining Staphylococcus aureus Cas9 (SaCas9) mRNA base editors for DSB-free knockout of B2M and REGNASE-1 with Streptococcus pyogenes Cas9 nucleases for targeted integration of an anti-CD19 CAR transgene at the T cell receptor α constant locus. Combined, these edits have been reported to generate safer allogeneic CAR-T cells with enhanced activity and persistence. We demonstrate multiplex gene editing in primary human T cells with B2M and REGNASE-1 base editing frequencies reaching 66% and 84%, respectively, while integrating the anti-CD19 CAR transgene in up to 36% or 71% of cells using nonviral single-stranded DNA repair templates or viral vector templates (AAV6), respectively. Importantly, no detrimental effects on CAR-T cell function were observed in vitro or in vivo, and knockout by base editing reduced rates of balanced chromosomal translocations by 210-fold. This orthogonal CRISPR-Cas engineering approach represents a novel and safer strategy for nonviral, multiplexed genetic engineering of CAR-T cells.