Abstract
There is a need of introducing large variant libraries into mammalian cells to facilitate deep mutational scanning (DMS). In this study, we develop an approach to integrate in-vitro ligation dsDNA variant library into cultured mammalian cell genome via the piggyBac transposon system. T4 DNA ligase was employed to produce dsDNA ligation products containing variant library sequence 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 results showed that about 29,000 resistance clones were generated from a single well of 6-well-plate transfected HEK293T cells. As a proof of principle of using the strategy for DMS, a GFP chromophore random variant library was integrated into the HEK293T cell genome. By FACS selection, we enriched GFP-positive cells. The next-generation sequencing (NGS) results showed that the GFP-chromophore amino acid sequences were restored after selection.
Competing Interest Statement
The authors have declared no competing interest.
