Non-viral targeted integration of large DNA in primary human T cells independent of double-stranded DNA breaks

Non-viral targeted integration of large DNA cargoes into human primary T cells typically requires the induction of genomic double-strand breaks (DSBs), a process associated with cytotoxicity and potential tumorigenic chromosomal abnormalities. Here we report PRIME-In, a novel genome-editing platform that uses a prime editing-engineered donor template coupled with either single (PRIME-In 1.0) or paired (PRIME-In 2.0) genomic nicks to enable precise integration of substantial DNA payloads into human cells without reliance on DSB repair pathways. Compared with traditional DSB-dependent methods, PRIME-In demonstrates markedly enhanced editing efficiency and specificity while eliminating detectable on-target and off-target chromosomal aberrations. Subsequent refinement of reagent composition and delivery protocols enabled PRIME-In-mediated engineering of primary human T cells with minimal toxicity, achieving up to 50% integration efficiency for a 3-kb CAR construct. These advances establish PRIME-In as a transformative platform for streamlining the non-viral production of genome-edited T cells, offering substantial potential for T cell-based immunotherapies.