CRISPR-based tools for targeted genetic manipulation in pathogenic species.

is an emerging fungal pathogen frequently associated with zoonotic transmission of sporotrichosis by contaminated cats. Within 25 years, the disease has spread not only throughout Brazil but now to neighboring countries in Latin America. Thermo-dimorphism, melanin, glycans, adhesins, and secreted vesicles have been associated with the ability of species to cause disease in the mammalian host. Although certain virulence factors have been proposed as potential determinants for sporotrichosis, the scarcity of molecular tools for performing reverse genetics in has significantly impeded the dissection of mechanisms underlying the disease. Here, we demonstrate that PEG-mediated protoplast transformation is a powerful method for heterologous gene expression in and . Combined with CRISPR/Cas9 gene editing, this transformation protocol enabled the deletion of the putative DHN-melanin synthase gene , which is a proposed virulence factor of species. To improve integration of deletion constructs, we deleted the KU homolog that is critical for non-homologous end-joining DNA repair. The use of Δ strains from enhanced homologous-directed repair during transformation resulting in increased targeted gene deletion in combination with CRISPR/Cas9. In conclusion, our CRISPR/Cas9-based transformation protocol provides an efficient tool for targeted gene manipulation in species. IMPORTANCE Sporotrichosis caused by is a disease that requires long periods of treatment and is rapidly spreading across Latin America. The virulence of this fungus and the surge of atypical and more severe presentations of the disease raise the need for an understanding of the molecular mechanisms underlying sporotrichosis, as well as the development of better diagnostics and antifungal therapies. By developing molecular tools for accurate genetic manipulation in this study addresses the paucity of reliable and reproducible tools for stable genetic engineering of species, which has represented a major obstacle for studying the virulence determinants and their roles in the establishment of sporotrichosis.