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

ABSTRACT Although the genus Coccidioides is divided into two closely related and putatively allopatric species, analysis shows that hybridization has occurred between species and at least one C. posadasii conserved fragment has introgressed into several C. immitis genomes in a population-specific manner. Transcript abundance in vitro and in vivo for 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 in Coccidioides pathogenesis. 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 in Coccidioides , and we employed the CRISPR-Cas9 mediated gene deletion tool to delete this gene in Coccidioides .