Repurposing base editors for targeted knockin and simultaneous multiplex knockouts to generate allo-CAR T cells with minimal translocations.

Multiplex genome editing of cellular therapies frequently requires multiple DNA double-strand breaks (DSBs), which can induce genotoxicity through chromosomal rearrangements and large deletions. Base editors enable targeted sequence changes with minimal DSBs and are widely used for gene disruption, but their capacity for transgene insertion has remained unexplored. Here, we have developed base editor-mediated knockin (BEKI), a non-viral platform combining transgene insertion with multiplex gene disruption using a single enzyme. BEKI repurposes the base editor's Cas9 nickase domain to generate paired nicks (inducing a localized DSB) at the knockin locus while achieving multiplex knockouts through base editing. Optimized guide RNA orientation and spacing enabled efficient transgene insertion across multiple T cell-relevant genomic loci. DNA-PK inhibition enhanced knockin efficiency but increased kilobase-scale deletions, which were mitigated by co-inhibition of Polθ. Compared with multiplex Cas9 editing, BEKI markedly reduced chromosomal translocations while preserving cell viability. BEKI supported targeted chimeric antigen receptor (CAR) knockin alongside up to 10 simultaneous gene knockouts, enabling the generation of allogeneic CAR T cells with enhanced cytokine secretion and resistance to immunosuppressants and allo-rejection. Together, BEKI provides a streamlined and scalable strategy for multiplex CAR T cell engineering with improved genomic stability, advancing safer next-generation cell therapies for cancer and autoimmune diseases.