Auxotrophic mutations of Trichophyton rubrum created by in vitro synthesized Cas9 ribonucleoprotein

Abstract

Background: 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. 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. Results: The 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. Conclusions:We 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.

Antifungal Activities of cis-trans Citral Isomers against Trichophyton rubrum with ERG6 as a Potential Target

“…A total of eight distinct enzymes participate in the conversion of lanosterol (the sterol) to ergosterol; however, most of these enzymes are membrane associated and unstable after isolation [23,24]. Further, target enzyme validation requires gene deletion experiments, which is a great challenge because only rudimentary protocols are presently available [25,26], compromising the development of novel ergosterol synthesis inhibitors. Recently, several studies reported that sterol pattern changes could be used as direct evidence to indicate that specific ergosterol biosynthesis enzymes are affected in several pathogenic fungi [27,28], which facilitate the identification of target enzymes after treatment with ergosterol synthesis inhibitors.…”

Targeting dermatophyte Cdc42 and Rac GTPase signaling to hinder hyphal elongation and virulence

“…14,15,17,64 Recently, auxotrophic mutants of T. rubrum have been generated using the Cas9 system. 65 In these articles, gene deletions and gene insertions were performed for functional analysis. Previous methods have made it difficult to analyze the functions of essential proteins that are potential drug targets.…”

Targeting dermatophyte Cdc42 and Rac GTPase signaling to hinder hyphal elongation and virulence

“… 14 , 15 , 17 , 67 , 68 Recently, auxotrophic mutants of T. rubrum have been generated using the Cas9 system. 69 In these studies, gene deletions and insertions were performed for functional analysis. Previous methods have made it difficult to analyze the functions of essential proteins that are potential drug targets.…”