Genomic integration of transgene by Bxb1 integrase

Abstract

Engineering cells to allow site-specific integration of large transgenes, such as synthetic genetic circuits, in a single-copy manner is believed to play a critical role especially in mammalian cells to dissect complex genetic networks and enact them with new functions for biomedical applications. We have tested and optimized a strategy for site-specific, single-copy integration of transgene into mammalian cells. The strategy involves the generation of a monoclonal chassis cell line to contain only one landing pad on one of the alleles using CRISPR-Cas9 techniques. The landing pad contains an attP site in which a plasmid containing an attB site could be integrated in the presence of Bxb1 integrase. The site-specific single-copy integration of transgene was validated. We demonstrated that a single monopartite nuclear localization signal (SV40NLS) positioned at the C terminus of Bxb1 produces higher integration efficiency than other Bxb1 variants harboring different types and positions of nuclear localization signal. Increasing the dosage of DNA plasmids and transfection reagent did not cause cytotoxicity but enhanced the transfection rate. Switching from FuGENE HD to another commercially available transfection reagent Lipofectamine 3000 produced transfection rate with less fluctuations along with enhanced integration efficiency. The approach in this study allows targeted integration of larger transgenes, such as synthetic circuits and combinatorial DNA libraries, in a single-copy manner into mammalian cell lines. This is valuable for research in functional assessment of large variant genetic libraries and multiplexed barcoded screenings. It would be useful if the maximum size of transgene to be integrated into the chassis cell can be tested in the future. This system could benefit a wider aspect of research including but not limited to mammalian cells when more cell types are engineered and tested as chassis cell lines.