Precision meets scale: DSB- and recombinase-free targeted insertion of large DNA fragments

Targeted insertion of large DNA fragments has been constrained by the genotoxicity associated with double-strand breaks (DSBs) and the limitations of two-step recombinase systems. Three independently developed mammalian platforms, Quadruple pegRNA (QuadPE), Prime Assembly (PA), and PRIME-In, now achieve programmable, DSB- and recombinase-free insertion of up to 26 kb, 11.3 kb, and 9.2 kb, respectively, through convergent yet mechanistically distinct flap-guided integration strategies. Notably, QuadPE and PA achieve integration independent of cell-cycle progression. Furthermore, these systems outperform several existing recombinase- and/or transposase-based tools, underscoring their translational potential for therapeutic utility. Their mechanistic logic also provides a forward path for the DSB-free and recombinase-free integration of large DNA sequences in crop biotechnology.