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Employing this sensitive assay, we revealed that the integration efficiencies of the 9.9-kb attP-containing DNA donor in rice cells remained remarkably high at 80.5%, relative to the 3.4-kb attP-containing donor, which was set as the baseline for 100% integration efficiency (Figure 2 B). However, a significant decrease in recombination efficiency was observed for donor DNA exceeding 17 kb. The integration efficiency of the 17.4-kb attP-containing plasmid decreased to 42.5% (Figure 2 B), and for the 27.3-kb attP-containing plasmid, it further declined to 8.2% (Figure 2 B). Similar trends were observed in Arabidopsis, with recombination efficiencies of 51.4%, 26.5%, and 9.0% for the 9.9-, 17.4-, and 27.3-kb attP-containing donors, respectively (Figure 2 C). Furthermore, PCR amplification of the attL junctions confirmed that the donor attP sequences had indeed been recombined into the attB as expected (Figures 2 D and 2E). Collectively, these results demonstrate that the Kp03 system can efficiently mediate targeted insertion of large DNA donors up to 27.3 kb in plant cells.

Interestingly, in addition to donor size, we also observed that Kp03-mediated recombination efficiency is sensitive to temperature, exhibiting differential efficiency under varying thermal conditions (Figure S2).