Cryptococcus genomic resources

点击翻译按钮获取中文摘要

Effective diagnosis and treatment of invasive fungal infections is a major unmet clinical challenge. Annotated genomic sequences of the major fungal pathogens create an opportunity to revolutionize medical mycology through the application of systematic approaches. To be maximally useful, genome sequences must be coupled with genome-wide biological resources. The availability of genome-wide knockout and tagged gene collections as well as a global genetic interaction map have been instrumental in catalyzing research progress in the model yeast Saccharomyces cerevisiae. The generation of analogous resources in pathogenic fungi creates experimental tools enabling comprehensive approaches to elucidating the basis of fungal virulence in the mammalian host and to develop drugs. Cryptococcus neoformans is an encapsulated budding yeast that causes fungal meningitis, resulting in ~200,000 fatalities each year. C. neoformans has haploid genetics, homologous recombination, and excellent animal models of disease. In the last period, we used biolistic transformation and long homology targeting constructs to complete a gene knockout collection, performed extensive quality control work, developed quantitative methods for screening the library in pools, and performed pooled screens both in mice and in vitro. We have made the strains available without restriction to the community via the Fungal Genetic Stock Center (FGSC). This resource has already been used in over 30 publications. To accelerate genome modification, we developed the first short-homology CRISPR-Cas9 methods for C. neoformans. We have also created an effective combined localization and purification tag. Preliminary work demonstrates that these methods can be deployed at scale to enable construction of a tagged strain collection. In addition, we have devised a novel method that enables pooled CRISPR-Cas9 screens in C. neoformans. Capitalizing on these technical advances, we will 1) complete the Cryptococcus whole-genome tag collection including production of an image resource, and 2) construct a first-generation genetic interaction map focused on virulence determinants. Accomplishment of the first aim will enable the cell biological and/or biochemical investigations of any protein. Completion of the second aim will deorphanize large fraction genes through membership in clusters of genes with known functions while defining new modules/pathways. This information will be integrated into the searchable and web accessible VEuPathDB database. Together these new genomic resources will accelerate the ability of the research community to understand and develop therapies against the most common cause of human fungal meningitis.