piggyBac-Mediated Genomic Integration of Linear dsDNA-Based Library for Deep Mutational Scanning in Mammalian Cells

Deep mutational scanning (DMS) makes it possible to perform massively parallel quantification of the relationship between genetic variants and phenotypes of interest. However, the difficulties in introducing large variant libraries into mammalian cells greatly hinder DMS under physiological states. Here we developed two novel strategies for DMS library construction in mammalian cells, namely 'piggyBac-in-vitro ligation' and 'piggyBac-in-vitro ligation-PCR'. For the first strategy, we took the 'in-vitro ligation' approach to prepare high-diversity linear dsDNAs, and integrate them into the mammalian genome with a piggyBac transposon system. For the second strategy, we further added a PCR step using the in-vitro ligation dsDNAs as templates, for the construction of high-content genome-integrated libraries via large-scale transfection. Both strategies could successfully establish genome-integrated EGFP-chromophore randomized libraries in HEK293T cells and enrich the green fluorescence-chromophore amino acid sequences. And we further identified a novel transcriptional activator peptide with the 'piggyBac-in-vitro ligation-PCR' strategy. Our novel strategies greatly facilitate the construction of large variant DMS library in mammalian cells, and may have great application potential in the future. Overall design: To introduce large variant libraries into the mammalian genome in a relatively simple way for DMS study, we revised our previously developed plasmid-independent library construction strategy called 'in-vitro ligation', a method that achieves transient library expression in mammalian cells via transfection of double-stranded DNAs (dsDNAs) with intact expression cassette which is obtained by ligation of PCR-generated dsDNAs containing degenerate codons. We sought to improve the 'in-vitro ligation' strategy by: (1) employing a piggyBac transposon system to integrate the in-vitro ligation dsDNAs into the mammalian genome, and (2) boosting the preparation yield of full-length in-vitro ligation dsDNAs via PCR reaction, thus facilitating large-scale transfection. Here we describe these improved strategies, which we refer to as 'piggyBac-in-vitro ligation' and 'piggyBac-in-vitro ligation-PCR' strategies, and validate their applications via the identification of green fluorescence-chromophore amino acid sequences and also a novel transcriptional activator peptide.