Abstract
There is a need to introduce large variant libraries into mammalian cells to facilitate deep mutational scanning (DMS). In this study, we develop approaches to integrate in-vitro ligation dsDNA variant library into cultured mammalian cell genome via the piggyBac transposon system. T4 DNA ligase was employed to generate dsDNA ligation products containing variant library sequences flanked by piggyBac inverted terminal repeat sequences (ITRs) at both ends. The ligation products were then cotransfected with a piggyBac transposase expression vector into mammalian cells for library integration. The clone formation assay result showed that about 29,000 resistance clones were generated from one single well of 6-well-plate transfected HEK293T cells. As a proof of the principle of using the strategy for DMS, a GFP chromophore random variant library was integrated into the HEK293T cell genome. By FACS selection, we collected the GFP-positive cells, and next-generation sequencing (NGS) showed that the GFP-chromophore amino acid sequences were enriched by the selection.
Competing Interest Statement
The authors have declared no competing interest.
