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Substrate Generation for Endonucleases of CRISPR/Cas Systems
Published on: September 8, 2012
Auxotrophic mutations of Trichophyton rubrum created by in vitro synthesized Cas9 ribonucleoprotein
Oliver Blechert1, Huan Mei1, Xiaohui Zang1
1Department of Medical Mycology, Institute of Dermatology, Chinese Academy of Medical Science and Peking Union Medical College, Nanjing, Jiangsu, 210042, People's Republic of China.
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.
Insights
We developed a new gene knock-out method for Trichophyton rubrum, a fungus causing most human skin infections. This method uses a Cas9-sgRNA system to enable genetic manipulation in this challenging pathogen.
Area of Science:
- Medical Mycology
- Molecular Biology
- Fungal Genetics
Background:
- Trichophyton rubrum is a major cause of human dermatomycosis, responsible for 80-90% of cases.
- Existing molecular genetic tools for T. rubrum are limited, hindering research and therapeutic development.
- Developing efficient genetic manipulation methods is crucial for understanding and combating this pathogen.
Purpose of the Study:
- To adapt and implement molecular genetic methods for targeted gene knockouts in Trichophyton rubrum.
- To establish a reliable protocol for gene manipulation in a non-model fungal pathogen.
- To overcome the challenges associated with genetic modification in T. rubrum.
Main Methods:
- Adaptation of gene knockout protocols from model fungal organisms.
- Utilizing in vitro synthesized Cas9-ribonucleoprotein complexes for targeted gene disruption.
- Development of a fungal transformation protocol involving PEG-mediated delivery into protoplasts.
- Selection of auxotrophic strains (e.g., URA3, TRP3) for subsequent genetic manipulations.
Main Results:
- Successful knockout of the URA3 gene, resulting in uracil auxotrophic T. rubrum strains.
- Demonstrated the ability to use these auxotrophic strains for further gene knockouts via marker reintegration.
- Exemplified the method by knocking out the TRP3 gene, producing tryptophan auxotrophic strains.
- Verified gene mutations through DNA sequencing.
Conclusions:
- A novel, efficient method for targeted gene knockouts in Trichophyton rubrum has been established using Cas9-sgRNA ribonucleoprotein complexes.
- The developed protocol enables the adaptation of sophisticated genetic manipulation techniques from model fungi to non-model pathogens like T. rubrum.
- This advancement facilitates deeper research into T. rubrum's pathogenicity and the development of new antifungal strategies.
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