PROJECT SUMMARY Advances in high throughput sequencing have already revealed millions of protein coding variants within human exomes, and there are many millions of additional differences that likely exist but have not yet been observed. Many of these variants likely play important roles influencing human health, but we lack the clinical data required to associate each variant genotype with their corresponding phenotypes. This disconnect is oftentimes referred to as the variant interpretation problem. Genetic experiments in model systems play a critical role in uncovering the effects of protein coding variants, but traditional approaches typically test variants one-by-one and will never address this glut of uncharacterized variants. Multiplex genetic assays capable of simultaneously testing complex variant libraries have the required throughput, but these approaches are still in their developmental infancy, and improvements are needed to increase the capabilities, costs, efficiency, and usability of these techniques to successfully address this problem. Harnessing a palette of synthetic biology tools centered around the highly efficient Bxb1 bacteriophage DNA recombinase, I developed a user-friendly, highly customizable platform for expression of complex variant libraries within individual cultured human cells. I previously paired this expression system with a highly generalizable assay that identifies variants that are loss-of-function due to an reduced intracellular steady-state abundance. I applied this assay to comprehensively study variants in four disease- related proteins, and more collaborative projects are still in progress. Unfortunately, these methods alone will not address the problem, and more orthogonal approaches are needed to tackle the millions of uncharacterized disease-relevant variants that exist within people. The goal of this proposal is to build the next set of fundamental biotechnologies needed to enable more high-throughput characterizations of protein variants. The individual directions described are each highly generalizable and can be reapplied to study large swaths of the proteome with only slight modification. Immediate directions include a functional complementation system to study essential genes, a fluorescent transcriptional reporter system to study perturbations to intracellular signaling pathways, and a barcoded ORFeome collection to identify genes that modulate phenotypes of interest when they are overexpressed. A major purpose of these technologies is to facilitate adoption by other research groups, especially those that are experts in other biological fields. These developments, along with the data we generate in the process of demonstrating their utility, will directly address the variant interpretation problem while also uncovering previously hidden biology underlying cancer-related molecular mechanisms critical to cell function.