Establishment of a CRISPR-Cas9 system for promoter recombination in cryptococcus deneoformans

Cryptococcus deneoformans is a basidiomycetous yeast found in multiple habitats across the globe. The species is a causative agent of cryptococcosis, a disease in which the inhalation of spores leads to fungal colonization of the lungs in various hosts. Cryptococcosis may also progress to other organs, notably the brain, which can result in cryptococcal meningoencephalitis. Unchecked cryptococcal disease is invasive, disruptive, and highly lethal, resulting in 13%-73% mortality rates despite treatment and 100% mortality when left untreated. Treatment options for cryptococcal disease are limited, especially in underdeveloped countries bearing the brunt of cryptococcal mortality due to lack of effective antifungal drugs. The search for drug targets in the C. neoformans species complex is being driven by molecular research into conserved proteins, many of which play key roles in cell survival and/or virulent processes. Studying the function of such proteins often requires complex genetic manipulations either in vitro or in vivo. This thesis aimed to test a CRISPR-Cas9 gene editing system in C. deneoformans that could be used to induce damage in precise gene targets, and also design a modular gene cassette that could recombine at those damaged sites by manipulating the endogenous DNA repair mechanisms of the cell. Editing was successfully accomplished in the URA5 gene by disrupting and later excising the coding region by homology-driven recombination with a modular gene cassette. In addition, the promoter region of the DNA polymerase gamma gene, MIP1, a putative essential gene, was replaced with a copper repressible promoter, CTR4p, to attempt to convey evidence for its necessity and suitability as a drug target