ArticleBMC biotechnology2020
Auxotrophic mutations of Trichophyton rubrum created by in vitro synthesized Cas9 ribonucleoprotein.
Article in BMC biotechnology, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 4 papers.
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4 citing papers in PubMed, 10 citations in OpenAlex.
- Targeting dermatophyte Cdc42 and Rac GTPase signaling to hinder hyphal elongation and virulence.iScience · 2024Article
- A Dual-Plasmid-Based CRISPR/Cas9-Mediated Strategy Enables Targeted Editing of pH Regulatory GeneJournal of fungi (Basel, Switzerland) · 2022Article
- Antifungal Activities ofMolecules (Basel, Switzerland) · 2021Article
- Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing.Theranostics · 2021Review
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Authors and funding
6 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
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.
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