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Efficient Generation of Multiple Seamless Point Mutations Conferring Triazole Resistance in Aspergillus fumigatus
Mariana Handelman1, Nir Osherov1
1Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Tel-Aviv 69978, Israel.
Abstract:
Aspergillus fumigatus is a common human fungal pathogen that can cause a range of diseases. Triazoles are used to treat A. fumigatus infections, but resistance is increasing due to mutations in genes such as cyp51A, hmg1 and overexpression of efflux pumps. Verifying the importance of these mutations is time-consuming, and although the use of CRISPR-Cas9 methods has shortened this process, it still relies on the construction of repair templates containing a selectable marker. Here, employing in vitro-assembled CRISPR-Cas9 along with a recyclable selectable marker, we devised a quick and easy way to effectively and seamlessly introduce mutations conferring triazole resistance in A. fumigatus. We used it to introduce, alone and in combination, triazole resistance-conferring mutations in cyp51A, cyp51B and hmg1. With the potential to seamlessly introduce genes imparting resistance to additional existing and novel antifungals, toxic metals, and environmental stressors, this technique can considerably improve the ability to introduce dominant mutations in A. fumigatus.
Insights
Researchers developed a faster CRISPR-Cas9 method to introduce drug resistance mutations in Aspergillus fumigatus, aiding the study of antifungal resistance. This technique streamlines the process of understanding how mutations in genes like cyp51A confer triazole resistance.
Area of Science:
- Mycology
- Molecular Biology
- Antimicrobial Resistance
Background:
- Aspergillus fumigatus is a significant human fungal pathogen.
- Increasing resistance to triazole antifungals is a major clinical challenge.
- Existing methods for studying resistance mutations are time-consuming.
Purpose of the Study:
- To develop a rapid and efficient method for introducing drug resistance mutations in A. fumigatus.
- To investigate the role of specific mutations in conferring triazole resistance.
- To create a versatile tool for studying gene function in A. fumigatus.
Main Methods:
- Utilized in vitro-assembled CRISPR-Cas9 technology.
- Employed a recyclable selectable marker for seamless gene editing.
- Introduced mutations in cyp51A, cyp51B, and hmg1 genes, individually and in combination.
Main Results:
- Successfully introduced triazole resistance-conferring mutations into A. fumigatus.
- Demonstrated the efficiency and speed of the new CRISPR-Cas9-based method.
- Validated the contribution of specific mutations to antifungal resistance.
Conclusions:
- The developed method offers a significant improvement for introducing dominant mutations in A. fumigatus.
- This technique can be adapted to study resistance to various agents, including antifungals and toxic metals.
- Facilitates faster research into fungal pathogen mechanisms and drug resistance.
