Matthew M. Morales

Coccidioides posadasii and C. immitis are human fungal pathogens endemic to the American Southwest. C. posadasii and C. immitis are the causative agents of coccidioidomycosis, or Valley fever. They are pathogens of growing concern, as reported cases of coccidioidomycosis have increased 20‐fold in the last two decades. Despite their importance as pathogens, Coccidioides spp . are understudied, especially compared to other human fungal pathogens with similar infectious burdens. The reasons Coccidioides spp . are understudied are multifactorial, including requirement of high biocontainment, technical difficulties in lab‐based culture growth, and a historic lack of genetic tools. Previous methods of genetic manipulation have been technically challenging, time consuming, and inefficient. Here, we present protocols for designing gene deletion constructs, generating protoplasts from both genetically engineered biosafety level 2 (BSL2) and wildtype biosafety level 3 (BSL3) strains, and performing transformations with CRISPR/Cas9. The protoplasting protocol described here uses a cell wall digestion enzyme employed in the wine‐making industry and results in high‐quality protoplasts that have the potential to be used for applications beyond transformations. We also present a high‐efficiency transformation method using CRISPR/Cas9. The protocols described here will allow for genetic manipulation of Coccidioides , using both BSL2 and BSL3 strains. This resource can be applied to expand research done in Coccidioides spp ., build molecular tools, and expand overall knowledge of these important pathogens. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Construction of gene deletion construct Alternate Protocol 1 : Construction of gene deletion construct Basic Protocol 2 : Creation of protoplasts in BSL3 Alternate Protocol 2 : Creation of protoplasts in BSL2 Basic Protocol 3 : CRISPR/Cas9 transformation in BSL3 Alternate Protocol 3 : CRISPR/Cas9 transformation in BSL2 Support Protocol : Passaging of mutants and PCR confirmation

The fungal disease Valley fever causes a significant medical and financial burden for affected people in the endemic region, and this burden is on the rise. Despite the medical importance of this disease, little is known about ecological factors that influence the geographic point sources of high abundance of the pathogens Coccidioides posadasii and C. immitis, such as competition with co-occurring soil microbes. These “hot spots”, for instance, those in southern Arizona, are areas in which humans are at greater risk of being infected with the fungus due to consistent exposure. The aim of this study was to isolate native microbes from soils collected from Tucson, Arizona (endemic area for C. posadasii) and characterize their relationship (antagonistic, synergistic, or neutral) to the fungal pathogen with in vitro challenge assays. Secreted metabolites from the microbes were extracted and described using analytical techniques including high-performance liquid chromatography (HPLC) and mass spectrometry. Bacteria belonging to the genus Bacillus and fungi in the Fennellomyces and Ovatospora genera were shown to significantly decrease the growth of Coccidioides spp. In vitro. In contrast, other bacteria in the Brevibacillus genus, as well as one species of Bacillus bacteria, were shown to promote growth of Coccidioides when directly challenged. The metabolites secreted from the antagonistic bacteria were described using HPLC and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The microbes identified in this study as antagonists to Coccidioides and/or the metabolites they secrete have the potential to be used as natural biocontrol agents to limit the amount of fungal burden at geographic point sources, and therefore limit the potential for human infection.