CRISPR/Cas9-Mediated Gene Disruption Reveals the Importance of Zinc Metabolism for Fitness of the Dimorphic Fungal Pathogen Blastomyces dermatitidis

Abstract

Blastomyces dermatitidis is a human fungal pathogen of the lung that can lead to disseminated disease in healthy and immunocompromised individuals. Genetic analysis of this fungus is hampered by the relative inefficiency of traditional recombination-based gene-targeting approaches. Here, we demonstrate the feasibility of applying CRISPR/Cas9-mediated gene editing to Blastomyces, including to simultaneously target multiple genes. We created targeting plasmid vectors expressing Cas9 and either one or two single guide RNAs and introduced these plasmids into Blastomyces via Agrobacterium gene transfer. We succeeded in disrupting several fungal genes, including PRA1 and ZRT1, which are involved in scavenging and uptake of zinc from the extracellular environment. Single-gene-targeting efficiencies varied by locus (median, 60% across four loci) but were approximately 100-fold greater than traditional methods of Blastomyces gene disruption. Simultaneous dual-gene targeting proceeded with efficiencies similar to those of single-gene-targeting frequencies for the respective targets. CRISPR/Cas9 disruption of PRA1 or ZRT1 had a variable impact on growth under zinc-limiting conditions, showing reduced growth at early time points in low-passage-number cultures and growth similar to wild-type levels by later passage. Individual impairment of PRA1 or ZRT1 resulted in a reduction of the fungal burden in a mouse model of Blastomyces infection by a factor of ~1 log (range, up to 3 logs), and combined disruption of both genes had no additional impact on the fungal burden. These results underscore the utility of CRISPR/Cas9 for efficient gene disruption in dimorphic fungi and reveal a role for zinc metabolism in Blastomyces fitness in vivo.

The Transcription Factor ZafA Regulates the Homeostatic and Adaptive Response to Zinc Starvation in Aspergillus fumigatus

“…We described for the first time that regulation of zinc homeostasis in a fungal pathogen is essential for virulence [ 13 , 14 ]. Similar studies, conducted later in different Candida and Cryptococcus species, Histoplasma capsulatum and Blastomyces dermatitidis , have also highlighted the importance of maintaining zinc homeostasis for the virulence of these pathogenic yeast [ 19 , 20 , 32 , 33 , 34 , 35 , 36 ]. In this paper, we report the transcriptional profiling of A. fumigatus under zinc starvation and show that ZafA regulates the expression of its most direct target genes through binding to one or more ZR consensus motifs located in their promoter regions.…”

Metabolism of Gluconeogenic Substrates by an Intracellular Fungal Pathogen Circumvents Nutritional Limitations within Macrophages

“…CRISPR/Cas9-mediated FBP1 gene knockout. Mutation of the FBP1 locus was done by CRISPR/ Cas9-mediated gene editing using a modification of a Cas9 and CRISPR guide RNA (gRNA) expression plasmid (53). The Cas9 and gRNA expression cassettes from pPTS608-Cas9-hyg were moved to a telomeric Histoplasma vector with a URA5 selection marker (pCR473 [54]).…”

The CRISPR toolbox in medical mycology: State of the art and perspectives

“…This review provides an updated overview of the state of the art of CRISPR-Cas9 genome editing in pathogenic fungi to both researchers and medical mycologists interested in using this technology. Though we focused on major fungal pathogens encountered in the clinical setting [102], CRISPR-Cas9 methods have also been developed in other human fungal pathogens not discussed here, such as Malassezia [103], Fusarium [104], and the dimorphic fungus Blastomyces dermatitidis [105]. The effectiveness and flexibility of CRISPR-Cas9 have the potential to have a dramatic impact on the field of medical mycology, both in the lab-by boosting genetic manipulation for studying fungal biology and pathogenesis, dissecting the role of virulence Table 1.…”