CRISPR/Cas9-Mediated Gene Disruption Reveals the Importance of Zinc Metabolism for Fitness of the Dimorphic Fungal Pathogen <named-content content-type="genus-species">Blastomyces dermatitidis</named-content>

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...

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Autores principales:	Gregory C. Kujoth, Thomas D. Sullivan, Richard Merkhofer, Taek-Jin Lee, Huafeng Wang, Tristan Brandhorst, Marcel Wüthrich, Bruce S. Klein
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	CRISPR fungi genetics pathogenesis virulence Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/e2b61624f02c4953831471247fdb9457
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spelling	oai:doaj.org-article:e2b61624f02c4953831471247fdb94572021-11-15T15:53:27ZCRISPR/Cas9-Mediated Gene Disruption Reveals the Importance of Zinc Metabolism for Fitness of the Dimorphic Fungal Pathogen <named-content content-type="genus-species">Blastomyces dermatitidis</named-content>10.1128/mBio.00412-182150-7511https://doaj.org/article/e2b61624f02c4953831471247fdb94572018-05-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00412-18https://doaj.org/toc/2150-7511ABSTRACT 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. IMPORTANCE Blastomyces is a human fungal pathogen that can cause serious, even fatal, lung infections. Genetic analysis of this fungus is possible but inefficient. We applied a recently developed gene editing technology, CRISPR/Cas9, to dramatically improve the efficiency with which gene disruptions are introduced into Blastomyces. We used this system to disrupt genes involved in zinc uptake and found that this reduced the fitness of the fungus upon infection.Gregory C. KujothThomas D. SullivanRichard MerkhoferTaek-Jin LeeHuafeng WangTristan BrandhorstMarcel WüthrichBruce S. KleinAmerican Society for MicrobiologyarticleCRISPRfungigeneticspathogenesisvirulenceMicrobiologyQR1-502ENmBio, Vol 9, Iss 2 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR fungi genetics pathogenesis virulence Microbiology QR1-502
spellingShingle	CRISPR fungi genetics pathogenesis virulence Microbiology QR1-502 Gregory C. Kujoth Thomas D. Sullivan Richard Merkhofer Taek-Jin Lee Huafeng Wang Tristan Brandhorst Marcel Wüthrich Bruce S. Klein CRISPR/Cas9-Mediated Gene Disruption Reveals the Importance of Zinc Metabolism for Fitness of the Dimorphic Fungal Pathogen <named-content content-type="genus-species">Blastomyces dermatitidis</named-content>
description	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. IMPORTANCE Blastomyces is a human fungal pathogen that can cause serious, even fatal, lung infections. Genetic analysis of this fungus is possible but inefficient. We applied a recently developed gene editing technology, CRISPR/Cas9, to dramatically improve the efficiency with which gene disruptions are introduced into Blastomyces. We used this system to disrupt genes involved in zinc uptake and found that this reduced the fitness of the fungus upon infection.
format	article
author	Gregory C. Kujoth Thomas D. Sullivan Richard Merkhofer Taek-Jin Lee Huafeng Wang Tristan Brandhorst Marcel Wüthrich Bruce S. Klein
author_facet	Gregory C. Kujoth Thomas D. Sullivan Richard Merkhofer Taek-Jin Lee Huafeng Wang Tristan Brandhorst Marcel Wüthrich Bruce S. Klein
author_sort	Gregory C. Kujoth
title	CRISPR/Cas9-Mediated Gene Disruption Reveals the Importance of Zinc Metabolism for Fitness of the Dimorphic Fungal Pathogen <named-content content-type="genus-species">Blastomyces dermatitidis</named-content>
title_short	CRISPR/Cas9-Mediated Gene Disruption Reveals the Importance of Zinc Metabolism for Fitness of the Dimorphic Fungal Pathogen <named-content content-type="genus-species">Blastomyces dermatitidis</named-content>
title_full	CRISPR/Cas9-Mediated Gene Disruption Reveals the Importance of Zinc Metabolism for Fitness of the Dimorphic Fungal Pathogen <named-content content-type="genus-species">Blastomyces dermatitidis</named-content>
title_fullStr	CRISPR/Cas9-Mediated Gene Disruption Reveals the Importance of Zinc Metabolism for Fitness of the Dimorphic Fungal Pathogen <named-content content-type="genus-species">Blastomyces dermatitidis</named-content>
title_full_unstemmed	CRISPR/Cas9-Mediated Gene Disruption Reveals the Importance of Zinc Metabolism for Fitness of the Dimorphic Fungal Pathogen <named-content content-type="genus-species">Blastomyces dermatitidis</named-content>
title_sort	crispr/cas9-mediated gene disruption reveals the importance of zinc metabolism for fitness of the dimorphic fungal pathogen <named-content content-type="genus-species">blastomyces dermatitidis</named-content>
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/e2b61624f02c4953831471247fdb9457
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CRISPR/Cas9-Mediated Gene Disruption Reveals the Importance of Zinc Metabolism for Fitness of the Dimorphic Fungal Pathogen <named-content content-type="genus-species">Blastomyces dermatitidis</named-content>

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