Basic enables selection-free efficient knockin of large DNA in primary human T cells

Efficient and precise insertion of large DNA fragments into primary human T cells has remained a bottleneck for gene and cell therapy. We present BaEVshort-AAV6 site-specific integration for CAR T (BASIC), a modular platform that combines BaEVshort-pseudotyped virus-like particles for Cas9 RNP delivery with AAV6 donor vectors for homology-directed repair. BASIC achieves >85% knockin efficiency without drug selection or electroporation, preserving cell viability while enabling multiplex genome engineering. Edited chimeric antigen receptor (CAR)-T cells show uniform CAR expression, enhanced cytotoxicity, and complete tumor clearance in vivo. BASIC offers a clinically scalable solution for next-generation cell therapies.