Next Article in Journal
A Dengue Vaccine: Will It be Accepted and Is It Feasible? Lessons from Barranquilla, Colombia, and Merida, Venezuela
Next Article in Special Issue
A Pyrrhic Victory: The PMN Response to Ocular Bacterial Infections
Previous Article in Journal
Gut Microbiome Modulation Based on Probiotic Application for Anti-Obesity: A Review on Efficacy and Validation
Previous Article in Special Issue
Current and Emerging Therapies for Ocular Herpes Simplex Virus Type-1 Infections
Open AccessArticle

CRISPR/Cas9-Mediated Gene Replacement in the Fungal Keratitis Pathogen Fusarium solani var. petroliphilum

1
Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
2
Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
*
Authors to whom correspondence should be addressed.
Microorganisms 2019, 7(10), 457; https://doi.org/10.3390/microorganisms7100457
Received: 13 September 2019 / Revised: 4 October 2019 / Accepted: 12 October 2019 / Published: 16 October 2019
(This article belongs to the Special Issue Insights Into The Molecular Pathogenesis of Ocular Infections)
Fungal keratitis (FK) is a site-threatening infection of the cornea associated with ocular trauma and contact lens wear. Members of the Fusarium solani species complex (FSSC) are predominant agents of FK worldwide, but genes that support their corneal virulence are poorly understood. As a means to bolster genetic analysis in FSSC pathogens, we sought to employ a CRISPR/Cas9 system in an FK isolate identified as Fusarium petroliphilum. Briefly, this approach involves the introduction of two components into fungal protoplasts: (1) A purified Cas9 protein complexed with guide RNAs that will direct the ribonuclease to cut on either side of the gene of interest, and (2) a “repair template” comprised of a hygromycin resistance cassette flanked by 40 bp of homology outside of the Cas9 cuts. In this way, Cas9-induced double strand breaks should potentiate double homologous replacement of the repair template at the desired locus. We targeted a putative ura3 ortholog since its deletion would result in an easily discernable uracil auxotrophy. Indeed, 10% of hygromycin-resistant transformants displayed the auxotrophic phenotype, all of which harbored the expected ura3 gene deletion. By contrast, none of the transformants from the repair template control (i.e., no Cas9) displayed the auxotrophic phenotype, indicating that Cas9 cutting was indeed required to promote homologous integration. Taken together, these data demonstrate that the in vitro Cas9 system is an easy and efficient approach for reverse genetics in FSSC organisms, including clinical isolates, which should enhance virulence research in these important but understudied ocular pathogens. View Full-Text
Keywords: Fusarium solani; Fusarium solani species complex; Fusarium petroliphilum; CRISPR/Cas9; genome editing; ura3 gene; 5-FOA Fusarium solani; Fusarium solani species complex; Fusarium petroliphilum; CRISPR/Cas9; genome editing; ura3 gene; 5-FOA
Show Figures

Figure 1

MDPI and ACS Style

Lightfoot, J.D.; Fuller, K.K. CRISPR/Cas9-Mediated Gene Replacement in the Fungal Keratitis Pathogen Fusarium solani var. petroliphilum. Microorganisms 2019, 7, 457.

Show more citation formats Show less citations formats
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Access Map by Country/Region

1
Back to TopTop