Analysis of the cyp51 genes contribution to azole resistance in Aspergillus section Nigri with the CRISPR-Cas9 technique.

Cyp51 contribution to azole resistance has been broadly studied and characterized in Aspergillus fumigatus, whereas it remains poorly investigated in other clinically relevant species of the genus, such as those of section Nigri In this work, we aimed to analyze the impact of cyp51 genes (cyp51A and cyp51B) on the voriconazole (VRC) response and resistance of Aspergillus niger and Aspergillus tubingensis We generated CRISPR-Cas9 cyp51A and cyp51B knock-out mutants from strains with different genetic backgrounds and diverse patterns of azole susceptibility. Single gene deletions of cyp51 genes resulted in 2 to 16-fold decrease of the VRC Minimum Inhibitory Concentration (MIC) values, which were below the VRC Epidemiological Cutoff Value (ECV) established by the Clinical and Laboratory Standards Institute (CLSI) irrespective of their parental strains susceptibilities. Gene expression studies in the tested species confirmed that cyp51A participates more actively than cyp51B in the transcriptional response of azole stress. However, ergosterol quantification revealed that both enzymes comparably impact the total ergosterol content within the cell, as basal and VRC-induced changes to ergosterol content was similar in all cases. These data contribute to our understanding on Aspergillus azole resistance, especially in non-fumigatus species.