Genome editing in Rhizopus delemar using using CRISPR-Cas systems

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Zygomycosis is the third most common invasive fungal infection that causes devastating diseases in individuals with immune deficiency, such as those with neutropenia or diabetic ketoacidosis. Mortality due to zygomycosis is very high, with a nearly 50% fatality rate even when appropriately treated. Due to the increasing prevalence of diabetic ketoacidosis, cancer, and organ transplantation, the number of patients at risk is dramatically increasing, such as in the case of a recent outbreak of hospital acquired Rhizopus delemar infection in New Orleans, which resulted in the death of at least six children. Despite such importance, very limited research resources are available to investigate R. delemar pathogenesis. This is partially due to the organism being not as genetically tractable as other pathogenic fungi. For example, there often exist multiple gene copies due to whole-genome duplication, and few mitotically stable genetic transformants can be obtained with the existing methodology. We propose to develop a CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) system that allows efficient gene specific manipulation and custom genome editing in R. delemar. We will begin by characterizing unique clinical strains in comparison to the archetypical strain for characteristics relevant to pathogenicity, auxotrophy, and resistance to antifungal compounds. We will construct vectors for Cas9 nuclease and guide RNA (gRNA) expression using the endogenous promoter and terminator sequences. We will then test the utility of the CRISPR-Cas system through manipulation of the marker PYRF (URA5) gene encoding orotidine phosphate decarboxylase, and the CNBR and CNA(A-C) genes encoding regulatory and catalytic subunits of calcineurin whose homologs play critical roles in fungal growth, morphogenesis, drug resistance, and pathogenesis. Successful implementation of our research plan will provide a revolutionary tool in promoting genetic studies of R. delemar pathogenesis mechanism.