Abstract
Transposases have transformed genetic engineering, yet functional systems remain scarce. In response, an unknown transposase system from Acyrthosiphon pisum was identified by metagenomic screening. Through systematic optimization, we enhanced nuclear localization, transposon architecture, and created a hyperactive transposase variant to boost efficiency. Intriguingly, the combined application of the newly discovered transposase with inverted terminal repeat sequences from a related pea aphid species, Aphis craccivora, further enhanced transposition activity, resulting in the first chimeric transposase system reported so far. We investigated the genomic integration events following transposition in mammalian cells to understand the underlying mechanisms and optimize the efficiency of transgene integration. This optimized system can expedite the generation of recombinant protein-producing Chinese Hamster Ovary (CHO) cell lines, even surpassing the hyperactive piggyBac system with regard to cell-specific productivity. These findings introduce a significant addition to the field of semi-targeted transgene integration technologies, offering substantial potential for enhancing biologics manufacturing.