A powerful and highly efficient PAI-mediated transgenesis approach in Drosophila.

We report a novel serine recombinase-mediated transgenesis approach in Drosophila melanogaster utilizing the Pseudomonasaeruginosa integrase (PAI), identified through a comprehensive bioinformatic analysis. PAI-mediated transgenesis achieves unparalleled integration efficiencies compared to the widely used PhiC31 system, with a 10-fold improvement in Drosophila S2 cells and transgenic efficiencies up to 61.9% in embryo microinjections, while exhibiting exceptional performance in integrating large transgenes up to 32 kb. We engineered versatile PAI-attP Drosophila lines spanning the three major chromosomes. Practical applications validate the utility and robustness of PAI-mediated transgenes. The PAI system's substantial advantages make it an invaluable tool for advancing Drosophila genetics, empowering high-throughput studies and novel disease modeling with unprecedented efficiency.