Abstract: The discovery of DNA variants implicated in genetic diseases is accelerating, but our interpretation of their function is currently trailing behind. This represents a significant limitation to the current utility of genetic data for patient diagnosis and stratification for therapy in diseases such as cancer. CRISPR technologies such as base editing facilitate the programmed installation of gene variants, allowing them to be directly coupled to their functional effect, helping to interpret the function of variants at scale. Here, I will describe the use of high-throughput base editing mutagenesis screens in cancer cells and tumour-derived organoids to aid interpretation of DNA variant function, with implications for precision medicine approaches. Furthermore, through a case study of base editing mutagenesis of JAK1 and the broader IFN-gamma signalling pathway, I will highlight how these functional genomics data can facilitate unbiased assessment of protein domain function, discovery of protein-protein interaction interfaces, and mechanisms of resistance to anti-tumour immunity. Functional annotation of mutations and their association with genetic diseases provides important supporting evidence for target identification. Moreover, mutagenesis screens can identify candidate protein domains or interaction interfaces for inhibition, without the need for in-depth structural analysis. Finally, I will describe how base editing mutagenesis screens can help verify drug mechanism of action and mechanisms of acquired resistance. Thus, these prospective mutagenesis screens have the potential to accelerate and inform the design of second-generation drugs for the effective treatment of therapy-resistant cancers.
