期刊
MBIO
卷 9, 期 2, 页码 -出版社
AMER SOC MICROBIOLOGY
DOI: 10.1128/mBio.00412-18
关键词
CRISPR; fungi; genetics; pathogenesis; virulence
类别
资金
- NIH [R01AI035681, R01AI040996, R21AI123758, T32AI055397]
- NIH National Institute on Minority Health and Health Disparities [F30 MD011547]
- American Heart Association [16POST30720006]
- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [R21AI123758, T32AI055397, R01AI035681, R01AI093553, R01AI130411, R01AI040996] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [T32GM008692] Funding Source: NIH RePORTER
- National Institute on Minority Health and Health Disparities [F30MD011547] Funding Source: NIH RePORTER
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 dualgene 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 similar to 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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据