show Abstracthide AbstractMultiplex genetic assays can simultaneously test thousands of genetic variants for a property of interest. However, limitations of existing multiplex assay methods in cultured mammalian cells hinder the breadth, speed, and scale of these experiments. Here, we describe a series of improvements that greatly enhance the capabilities of a Bxb1 recombinase-based landing pad system for conducting different types of multiplex genetic assays in various mammalian cell lines. We incorporate the landing pad into a lentiviral vector, easing the process of generating new landing pad cell lines. We also develop several new landing pad versions, including one where the Bxb1 recombinase is expressed from the landing pad itself, improving recombination efficiency more than 2-fold and permitting rapid prototyping of transgenic constructs. Other versions incorporate positive and negative selection markers that enable drug-based enrichment of recombinant cells, enabling the use of larger libraries and reducing costs. A version with dual convergent promoters allows enrichment of recombinant cells independent of transgene expression, permitting the assessment of libraries of transgenes that perturb cell growth and survival. Finally, we demonstrate these improvements by assessing the effects of a combinatorial library of oncogenes and tumor suppressors on cell growth. Collectively, these advancements make multiplex genetic assays in diverse cultured cell lines easier, cheaper and more effective, facilitating future studies probing how proteins impact cell function, using transgenic variant libraries tested individually or in combination. Overall design: We created a molecular cloning framework that allows for combinatorial barcoding, expression, and sequencing of two distinct transgenes separated by a 2A element. Each transgene in each position was given a 16 nucleotide identifier sequence, separated by a 20 nucleotide constant region. Each 16 nucleotide identifier was composed of eight nucleotides of known sequence serving as a transgene identifier, flanked on either side by four degenerate nucleotides serving as unique plasmid identifier sequences. The plasmid preparation was sequenced in two seperate instances. We recombined the library into LLP-rEF1a cells (Fig 3a) and grew the cells for two weeks, enricheding recombined cells to ~40%. Genomic DNA was extracted from each cell pellet, and recombined plasmid barcodes were amplified and sequenced at high throughput. We performed a total of eight replicate experiments from two independent recombinations.