Exploring Different Approaches for Constructing Temperature Sensitive Alleles of Yeast Essential Genes

Exploring the phenotypes associated with conditional alleles of essential genes has been widely used to study gene function in multiple model systems. In the budding yeast, Saccharomyces cerevisiae, geneticists have long made use of temperature-sensitive (TS) alleles, which support cell growth at permissive but not at non-permissive temperatures, to study essential gene function. Over the years, TS mutants have been isolated in most yeast essential genes, but a subset has been refractory to standard methods for isolating TS mutants. In my thesis project, I aimed to explore new and existing methods for systematic gene mutagenesis, with the goal of completing the TS collection of yeast essential gene mutants. I also aimed to use the TS collection as a resource to explore mechanisms that underly the phenomenon of temperature sensitivity.To approach my goals, I explored two different methods, deactivated Cas9 linked to Cytidine Deaminase and the CRISPEY mutagenesis system, in both a low and high throughput manner, to determine which method may be most suitable for construction of new TS alleles of yeast essential genes. I also analyzed existing TS mutations to better understand what mutations give rise to temperature sensitivity, which may guide construction of TS alleles in other genes.M.Sc