A singular base-editing platform for polyfunctional multiplex engineering of immune cells.

Current methods to engineer antigen-specific receptors rely on randomly integrating vectors or double-strand-break-induced targeted integration, both of which pose safety risks. To implement an all-in-one tool for multiplex knockout (KO) and knockin (KI), we expand the use of base editor nickase activity to stimulate homology-directed repair (HDR) and insert clinically relevant chimeric antigen receptors (CARs) into specific loci. Through a novel single-guide RNA design strategy and a DNA template delivered by a recombinant adeno-associated virus, we enhanced the efficiency of ABE8e-stimulated HDR in human T cells. By combining KI of CD19, CD33, or mesothelin-targeting CARs with >95% quadplex gene KO (B2M/CD3E/PDCD1/CISH), we achieve single-step generation of highly functional off-the-shelf CAR T cell products with enhanced function. Importantly, we found no detectable translocations or significant off-target edits and demonstrated efficacy against multiple cancer lines and a suppressive 3D spheroid culture model. This efficient engineering process of "Iterative Nicking for Synchronous Engineered Reprogramming of T cells" (INSERT) establishes a safe, simplified platform for advanced therapeutic CAR T engineering.