Validation of candidate genes for drug resistance in model organism Saccharomyces cerevisiaeŠkofljanc, Neža (Avtor) Petrovič, Uroš (Mentor) Mozzachiodi , Simone (Komentor) Fungal infectionsantifungal drug resistanceSaccharomyces cerevisiaeGWASCRISPR-Cas9stress responseAntibiotic resistance is an increasing threat worldwide due to the high number of microbes developing resistance to the limited range of drugs. Yeasts, as other microbes, can develop antifungal resistance when exposed to these compounds and produce difficult to treat infections. In this work we took advantage of a set of 420 genetic variants that have been mapped as genetic determinants in regulating antifungal resistance in a large cohort of Saccharomyces cerevisiae strains. We looked into those variants and chose the most compelling ones, which map onto four different genes – HSP82, AIM29, PIS1 and FKS1. We applied the CRISPR-Cas9 technology to engineer those variants into genetically different S. cerevisiae strains. Then we tested the constructed strains for their response to two different antifungal drugs, ketoconazole and fluconazole. The tested phenotypes were generation time (i.e., growth speed) and yield (i.e., final population density) in the presence of the drug compared to the drug-free medium. All of the inspected variants, in at least one genetic background, caused a change in drug sensitivity in the engineered strains compared to the parent strains. Furthermore, we measured the growth of the mutants at two different temperatures, 30 °C and 37 °C, to inspect whether the mutations improve or impair the growth of the strains at elevated temperature but saw no such effect. Lastly, we phenotyped the constructed strains for their chronological lifespan and measured a marked increase in the strains with altered variants in AIM29 and FKS1 genes.20232023-04-19 07:16:05Magistrsko delo/naloga145350sl