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Development of CRISPR-Cas9 genome editing system in Talaromyces marneffei
Xiangmei Zhang1, Xueyan Hu2, Saad Jan3
1Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, 100191, China.
Abstract:
Talaromyces marneffei is an important pathogenic thermally dimorphic fungus causing systemic talaromycosis mainly prevalent in Southeast Asia. The dimorphic transition between mycelium and yeast is considered crucial for the pathogenicity of T. marneffei. However, the lack of genetic toolbox has been a major impediment for understanding its pathogenicity. Here a CRISPR-Cas9 system was developed to facilitate genetic manipulations in this organism. In this study, the CRISPR-Cas9 gene editing system uses a native U6 snRNA promoter from T. marneffei to drive the expression of sgRNA. Employing this system and PEG-mediated protoplast transformation, the sakA gene was mutated. Sanger sequencing confirmed nearly 40% site-directed mutation rate. The phenotype analysis confirmed the sakA gene function in T. marneffei dimorphic transition. Our study provided a powerful genome-manipulating tool, which could accelerate studies on T. marneffei for further revealing the mechanisms of its pathogenicity.
Insights
Researchers developed a CRISPR-Cas9 gene editing tool for Talaromyces marneffei, a fungus causing talaromycosis. This new system enables genetic manipulation, aiding in understanding fungal pathogenicity and dimorphic transition.
Area of Science:
- Medical Mycology
- Molecular Biology
- Fungal Genetics
Background:
- Talaromyces marneffei causes systemic talaromycosis, a significant fungal infection in Southeast Asia.
- The fungus's dimorphic transition (mycelium to yeast) is critical for its pathogenicity.
- Limited genetic tools have hindered research into T. marneffei's virulence mechanisms.
Purpose of the Study:
- To develop a CRISPR-Cas9 gene editing system for Talaromyces marneffei.
- To enable efficient genetic manipulation in this pathogenic fungus.
- To investigate the role of specific genes, like sakA, in T. marneffei's biology.
Main Methods:
- A CRISPR-Cas9 system was engineered using a native U6 snRNA promoter from T. marneffei.
- The system was utilized with PEG-mediated protoplast transformation for gene editing.
- The sakA gene was targeted for mutation, and outcomes were verified by Sanger sequencing.
Main Results:
- A CRISPR-Cas9 system was successfully established for T. marneffei, facilitating genetic manipulation.
- A site-directed mutation rate of approximately 40% was achieved for the sakA gene.
- Phenotypic analysis confirmed the sakA gene's essential role in the dimorphic transition of T. marneffei.
Conclusions:
- The developed CRISPR-Cas9 system provides a powerful tool for genome manipulation in T. marneffei.
- This advancement will accelerate research into the pathogenicity mechanisms of T. marneffei.
- Understanding the sakA gene's function offers insights into fungal dimorphism and virulence.
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