PROJECT SUMMARY/ABSTRACT Little is known about virulence mechanisms for most emerging fungal pathogens. Coccidioides immitis and C. posadasii, are of specific concern due to rapid increase in disease (coccidioidomycosis, aka Valley fever) incidence in the US, with a more than 10-fold increase in reported disease over the last 20 years. The Centers for Disease Control and Prevention (CDC) report that average cost for treating Valley fever is $50,000 per patient, including extensive hospital stays and treatment regimes. Use of CRISPR/Cas9 and functional assessment of targeted gene deletion on virulence can help improve our understanding of mechanisms of pathogenicity and represent the foundational framework that is necessary for development of these approaches. We propose to functionally delete 10 gene targets in Coccidioides posadasii strain Silveira and assay these deletions for loss of virulence. We will further investigate pathogenicity in C. immitis strain RS for those genes that show a loss of virulence. Currently, few specific mechanisms of virulence have been investigated in Coccidioides. Analysis of these data will provide new insights into functions of Coccidioides genes, which will increase our understanding of fungal pathogenesis mechanisms. Finally, we will work to develop tools to engineer fluorescently labeled live strains for Coccidioides using mCherry expression that will increase tools for interrogating host:pathogen interactions in an in vitro cell culture assay. In total, this proposal represents needed improvements in functional assessment of virulence in an important, yet understudied, fungal pathogen.