Reconstitution of Prospermatogonial Specification in Vitro from Human Induced Pluripotent Stem Cells
Mammalian Synthetic Biology Workshop
2020
Virtual International Mammalian Synthetic Biology Workshop (mSBW) 2020
General Submissions
General Submission (Oral)
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.