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Generation of Seamless Point Mutations with Cas9 RNP and pTel-hygR Plasmid in Aspergillus fumigatus
Mariana Handelman1, Hila Werner1, Nir Osherov2
1Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Tel-Aviv, Israel.
Abstract:
The environmental mold Aspergillus fumigatus is a common human fungal pathogen that causes a wide range of diseases. The antifungal triazoles that inhibit the Cyp51 enzyme involved in ergosterol biosynthesis are used to treat A. fumigatus infections. However, triazole resistance is an increasing concern due to mutations in the genes cyp51A, hmg1, and others, and efflux pumps overexpression. The process of verifying mutations is time-consuming, even with CRISPR-Cas9 methods, as it still requires constructing repair templates with selectable markers. This study presents a faster and more efficient method to introduce mutations conferring triazole resistance in A. fumigatus by using in vitro assembled CRISPR-Cas9, along with a recyclable selectable marker. With this approach, we successfully introduced triazole resistance-conferring mutations in A. fumigatus genes (cyp51A, cyp51B, and hmg1), both individually and in combination. The technique has the potential to introduce mutations for resistance to other antifungals, toxic metals, and environmental stressors, thus enhancing the ability to generate dominant mutations in A. fumigatus.
Insights
Researchers developed a faster CRISPR-Cas9 method to create drug resistance mutations in Aspergillus fumigatus. This technique aids in studying fungal pathogens and developing new antifungal treatments.
Area of Science:
- Mycology
- Molecular Biology
- Antimicrobial Resistance
Background:
- Aspergillus fumigatus is a common fungal pathogen causing various diseases.
- Triazoles are key antifungals targeting ergosterol biosynthesis but face resistance.
- Existing mutation verification methods are time-consuming, hindering research.
Purpose of the Study:
- To develop a faster and more efficient method for introducing drug resistance mutations in A. fumigatus.
- To utilize in vitro assembled CRISPR-Cas9 with a recyclable marker for precise gene editing.
- To generate mutations conferring triazole resistance in key A. fumigatus genes.
Main Methods:
- Employed in vitro assembled CRISPR-Cas9 system for targeted gene modification.
- Integrated a recyclable selectable marker to streamline the mutation introduction process.
- Successfully introduced mutations in cyp51A, cyp51B, and hmg1 genes, individually and in combination.
Main Results:
- Demonstrated a significantly faster and more efficient method for generating triazole resistance mutations.
- Successfully created single and combined mutations in cyp51A, cyp51B, and hmg1 genes.
- Validated the versatility of the technique for introducing various resistance-conferring mutations.
Conclusions:
- The novel CRISPR-Cas9 approach accelerates the generation of resistant A. fumigatus strains.
- This method enhances the study of antifungal resistance mechanisms and the development of new therapies.
- The technique holds potential for generating mutations conferring resistance to diverse environmental challenges.
