Mammalian Genomic Manipulation with Orthogonal Bxb1 DNA Recombinase Sites for the Functional Characterization of Protein Variants

Abstract

The Bxb1 bacteriophage serine DNA recombinase is an efficient tool for engineering recombinant DNA into the genomes of cultured cells. Generally, a single engineered “landing pad” site is introduced into the cell genome, permitting the integration of transgenic circuits or libraries of transgene variants. While sufficient for many studies, the extent of genetic manipulation possible with a single recombinase site is limiting and insufficient for more complex cell-based assays. Here, we harnessed two orthogonal Bxb1 recombinase sites to enable alternative avenues for using mammalian synthetic biology to characterize transgenic protein variants. By designing plasmids flanked by a second pair of auxiliary recombination sites, we demonstrate that we can avoid the genomic integration of undesirable bacterial DNA elements using the same starting cells engineered for whole-plasmid integration. We also created “double landing pad” cells simultaneously harboring two orthogonal Bxb1 recombinase sites at separate genomic loci, allowing complex cell-based genetic assays. Integration of a genetically encoded calcium indicator allowed for the real-time monitoring of intracellular calcium signaling dynamics, including kinetic perturbations that occur upon overexpression of the wild-type or variant version of the calcium signaling relay protein STIM1. A panel of missense mutants of the HIV-1 accessory protein Vif was paired with various paralogs within the human Apobec3 innate immune protein family to identify combinations capable or incapable of interacting within cells. These cells allow transgenic protein variant libraries to be readily paired with assay-specific protein partners or biosensors, enabling new functional readouts for large-scale genetic assays for protein function.

Increased stable integration efficiency in CHO cells through enhanced nuclear localization of Bxb1 serine integrase

“…Bxb1 integrase has been extensively used for the stable genomic integration of protein variant libraries into mammalian cells [ 7 , 8 , 10 , 11 , 23 , 24 ]. The likelihood of finding a protein variant with desired properties rises with the expansion of the variant library size.…”