Novel allogeneic CAR T-cell platform involving microhomology-mediated end joining repair and low off-targeting potential

Several allogeneic chimeric antigen receptor (CAR) T-cell therapies in clinical trials rely on CRISPR-Cas genome editing, but the enzyme's random repair mechanism increases the risk of undesired off-target effects, challenging safe CAR T-cell generation. To address this, we developed a novel CRISPR RNA (crRNA) targeting the T-cell receptor beta constant (TRBC) gene. Combined with AsCas12a Ultra, this crRNA edits primary human T-cells via a predictable microhomology-mediated end joining (MMEJ) DNA repair pathway, significantly lowering off-target risks. During evaluation, we sequestered a unique T-cell subset with disrupted T-cell receptor (TCR), retained CD3 expression, and no <i>in vivo</i> alloreactivity. Termed CD3-retained, allogeneically functioning T-cells (CRAFT-cells), these cells exhibited growth kinetics comparable to unedited T-cells. When engineered with CD19- or BAFF-R-targeted CARs, CRAFT CAR T-cells showed strong antigen-specific cytotoxicity and significant <i>ex vivo</i> expansion compared to conventional CD3-disrupted CAR T-cells. Moreover, CRAFT CAR T-cells effectively served as effector cells for bispecific T-cell engagers (BiTEs), enabling CD3-dependent tumor cell killing. Our CRAFT crRNA platform offers a novel strategy to generate safer allogeneic CAR T-cells. The distinct properties of CRAFT CAR T-cells, combined with BiTE therapy, represent a promising and potentially more durable approach for next-generation allogeneic CAR T-cell therapies in clinical applications.