High-Efficiency System for Site-Specific Transgene Integration in Mammalian Cells
Mammalian Synthetic Biology Workshop
The ability to insert a transgenic payload into a specific locus in a host cellâs chromosome facilitates the creation of cell lines that reliably express therapeutic transgenes or synthetic gene circuits. Such targeting can be achieved with bacteriophage-derived serine integrases â enzymes that catalyze recombination between short DNA sequences, called attP and attB sites. Previous work has shown that the Bxb1 integrase works efficiently in mammalian cells. Here, we show that a point mutation in the Bxb1 attP and attB sites, termed Bxb1-GA, nearly doubles the efficiency of Bxb1-mediated integration, resulting in the highest integration efficiency reported to date in mammalian cells. Bxb1-GA sites and wild-type Bxb1 sites do not cross-react, and hence can be used in parallel for orthogonal targeting of distinct payloads to different sites. Our data present an improved tool for site-specific transgene integration in mammalian cells for research and biomanufacturing purposes.