Using double cutin vitroassembled CRISPR/Cas9 to modify the genome ofCoccidioides posadasii

Although the genusCoccidioidesis divided into two closely related and putatively allopatric species, analysis shows that hybridization has occurred between species and at least oneC. posadasiiconserved fragment has introgressed into severalC. immitisgenomes in a population-specific manner. Transcript abundancein vitroandin vivofor ten ORFs in this introgressed region were measured for several isolates. We used signals of introgression and high mRNA transcript levels in the spherule as indicators of selection for genes related to critical biological processes involved inCoccidioidespathogenesis. The only transcript in the introgression region with significant expression was a gene that encodes for a betadefensin-like (DEFBL) peptide rich in serines and cysteines. Few virulence factors have been identified inCoccidioides, and we employed the CRISPR-Cas9 mediated gene deletion tool to delete this gene inCoccidioides.