Raw ddPCR and FACS data for "Non-viral targeted integration of large DNA in primary human T cells independent of double-stranded DNA breaks"

Conventional non-viral strategies for targeted integration of large, diverse DNA cargoes into human primary T cells typically rely on the induction of genomic double-strand breaks (DSBs)—a process associated with genotoxic effects and potential tumorigenic chromosomal abnormalities. Herein, we present PRIME-In (Prime Editing-Mediated Large Integration), a novel genome-editing platform that employs 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 to traditional DSB-dependent methods, PRIME-In demonstrates markedly enhanced editing efficiency and specificity while eliminating detectable chromosomal aberrations at both on-target and predicted off-target loci. Subsequent refinement of reagent composition and delivery protocols enabled PRIME-In-mediated engineering of primary human T cells with minimal toxicity, achieving integration efficiencies of up to 60%. Furthermore, this platform facilitated the generation of functional chimeric antigen receptor (CAR) T cells with integration efficiencies up to 30%, yielding a 30-fold expansion of T cell populations within 7 days of ex vivo culture. These developments suggest that PRIME-In could serve as a useful platform for simplifying the non-viral generation of genome-edited T cells, potentially providing opportunities to improve manufacturing approaches for emerging immunotherapies.