Genetic and chemo-genetic interactions have played key roles in elucidating the molecular mechanisms by which certain chemicals perturb cellular functions. Many studies have employed gene knockout collections or gene disruption/depletion strategies to identify routes for evolving resistance to chemical agents. By contrast, searching for point-mutational genetic suppressors that can identify separation- or gain-of-function mutations, has been limited even in simpler, genetically amenable organisms such as yeast, and has not until recently been possible in mammalian cell culture systems. Here, by demonstrating its utility in identifying suppressors of cellular sensitivity to the drugs camptothecin or olaparib, we describe an approach allowing systematic, large-scale detection of spontaneous or chemically-induced suppressor mutations in yeast and in haploid mouse embryonic stem cells in a short timeframe, and with potential applications in essentially any other haploid system. In addition to its utility for molecular biology research, this protocol can be used to identify drug targets and to predict mechanisms leading to drug resistance. Mapping suppressor mutations on the primary sequence or three-dimensional structures of protein suppressor hits provides insights into functionally relevant protein domains, advancing our molecular understanding of protein functions, and potentially helping to improve drug design and applicability
