Multiplex Functional Characterization of Protein Variant Libraries in Mammalian Cells with Single-Copy Genomic Integration and High-Throughput DNA Sequencing

Abstract

Sequencing-based, massively parallel genetic assays have enabled simultaneous characterization of the genotype-phenotype relationships for libraries encoding thousands of unique protein variants. Since plasmid transfection and lentiviral transduction have characteristics that limit multiplexing with pooled libraries, we developed a mammalian synthetic biology platform that harnesses the Bxb1 bacteriophage DNA recombinase to insert single promoterless plasmids encoding a transgene of interest into a pre-engineered "landing pad" site within the cell genome. The transgene is expressed behind a genomically integrated promoter, ensuring only one transgene is expressed per cell, preserving a strict genotype-phenotype link. Upon selecting cells based on a desired phenotype, the transgene can be sequenced to ascribe each variant a phenotypic score. We describe how to create and utilize landing pad cells for large-scale, library-based genetic experiments. Using the provided examples, the experimental template can be adapted to explore protein variants in diverse biological problems within mammalian cells.

Keywords: Bxb1 integrase; DNA recombinase; Deep mutational scanning; Functional genetic experiments; High-throughput DNA sequencing; Landing pad; Multiplex assays of variant effect.