Abstract
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.
Highlights
The transparency of the cornea—due to its avascularity, paucity of resident cells, and careful assembly of collagen fibrils—allows for the entry of light into the eye for proper vision [1]
All experiments were conducted with a clinical fungal keratitis (FK) isolate (06-0110) from UCSF, previously identified as Fusarium solani species complex (FSSC) based on internal transcribed spacer (ITS) sequencing [28]
Strains were cultured on glucose minimal medium (GMM) (1% glucose, clutterbuck salts, hunters trace elements, pH 6.5) or yeast, peptone, and dextrose medium (YPD; 2% dextrose, 2% peptone, 1% yeast extract) at 30 ◦C. 5 mM of uracil and uridine were added when specified. 2 g/L of 5-fluorotic acid (FOA) was used in specified transformation selection media, and 1 g/L was used for further phenotyping [30]
Summary
The transparency of the cornea—due to its avascularity, paucity of resident cells, and careful assembly of collagen fibrils—allows for the entry of light into the eye for proper vision [1]. In south India, for example, F. falciforme accounts for ~80% of all FSSC keratitis cases [18] This organism is the most common Fusarium species isolated from soil and vegetation, which fits with FK resulting from agricultural-related trauma [19]. We further demonstrate that the use of CRISPR increases the transformation yield overall, providing more colonies that can be screened and increasing the likelihood of isolating the desired mutant Taken together, this methodology represents a simple and efficient means for targeted gene replacement in a clinical FSSC isolate, which should bolster reverse genetic studies in these important but understudied pathogens
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