Generation of Seamless Point Mutations with Cas9 RNP and pTel-hygR Plasmid in Aspergillus fumigatus.

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.