Abstract
BackgroundTrichophyton rubrum is an obligate human parasitic fungus and responsible for approximately 80–90% of dermatomycosis in human. Molecular genetic manipulations of this pathogen are challenging and available tools and protocols are only rudimentary. We adapt molecular genetics methods of well established fungal model organism, to knock out genes in T. rubrum. For the adaptation, crucial modifications are necessary. With the implementation of in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, it is possible to adapt molecular genetic methods, to knock out genes in T. rubrum.ResultsThe gene knock-out method is based on integration of a selection marker into the target site, to interrupt the gene translation. The target gene gets preassigned by the homologous sequence of the in vitro synthesized Cas9-sgRNA ribonucleoprotein complex. To develop the method, we first isolated and characterized a T. rubrum strain with a high amount of microconidia. Next, we developed a transformation protocol, whereby the Cas9-sgRNA ribonucleoprotein gets delivered into the fungal protoplast by the PEG method.We knocked out the URA3 gene and resulted, as predicted, uracil auxotrophic strains. These strains can be used for specific gene knock-outs by reintegrating the URA3 fragment and selection on uracil lacking cultivation media. Exemplary, we knocked out the TRP3 gene and got the predicted phenotype, tryptophan auxotrophic strains. The mutation had been verified by sequencing.ConclusionsWe developed a method, based on in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, for target specific gene knock-outs in T. rubrum. We knocked out the Ura3 gene and resulted uracil auxotrophic strains. These strains were used for target specific gene knock-outs by reintegrating the Ura3 fragment into the target gene site to interrupt the gene transcription. The developed method allows to adapt sophisticate gene manipulation methods of model fungal species to non-model species.
Highlights
Trichophyton rubrum is an obligate human parasitic fungus and responsible for approximately 80–90% of dermatomycosis in human
We developed for the first time a transformation system for T. rubrum, based on delivering the Protein of the CRISPR/Cas9 system (Cas9)-sgRNA ribonucleoprotein complex together with the Orotidine-5′-monophosphate decarboxylase gene (URA3) gene DNA fragment into the protoplast by the PEG method
The fungi were identified by phenotype, microscopic analysis and comparison of the internal transcribed spacer (ITS) sequence
Summary
Trichophyton rubrum is an obligate human parasitic fungus and responsible for approximately 80–90% of dermatomycosis in human. Molecular genetic manipulations of this pathogen are challenging and available tools and protocols are only rudimentary. We adapt molecular genetics methods of well established fungal model organism, to knock out genes in T. rubrum. With the implementation of in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, it is possible to adapt molecular genetic methods, to knock out genes in T. rubrum. As an obligate human pathogenic fungus, T. rubrum is in the focus of medicine and research for more than a century. In fungal model-organism auxotrophic selection-markers are a standard tool for selecting transformed colonies. A special advantage of using the URA3 gene as (2020) 20:6 selection-marker is the possibility of counter-selection by 5fluoroorotic acid (FOA) [2]. S. cerevisiae and C. albicans Δura strains can not grow in the absence of uracil, but can grow on FOA media, whereas the wild type can grow in the absence of uracil but not on FOA media
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