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OBJECTIVES: Candidozyma (Candida) auris is an emerging fungal pathogen of global concern that often exhibits multi-drug resistance. Of the six described C. auris clades, Clade III has been found to be nearly universally fluconazole-resistant, and almost every Clade III isolate described carries a mutation leading to a VF125AL substitution in ERG11, the gene encoding the fluconazole target lanosterol 14α-demethylase, and a mutation leading to a N647T substitution in the gene encoding Mrr1a, a transcriptional regulator of the Mdr1 transporter. Here we aimed to determine the specific contributions of these mutations to triazole antifungal susceptibility in Clade III C. auris isolates.METHODS: We introduced the Clade III MRR1A mutation into a Clade I background using CRISPR-Cas9 gene editing. In two Clade III clinical isolates we corrected the native MRR1A and ERG11 mutations to their wild-type sequences as well as disrupted MDR1. Triazole susceptibilities and MDR1 gene expression were measured in all strains.RESULTS: Introduction of the N647T substitution in a Clade I background increases fluconazole MIC from 2 to 4 μg/mL. Correction of MRR1A or disruption of MDR1 in each Clade III background resulted in a one-dilution decrease in fluconazole MIC (from 64 to 32 μg/mL) while the ERG11 correction resulted in a three- to four-dilution decrease (from 64 to 4-8 μg/mL) in fluconazole MIC. Correction of both mutations together resulted in a further decrease to 4 μg/mL. Similar changes were observed for voriconazole.CONCLUSIONS: Our findings show that while the MRR1A mutation makes a modest contribution, the ERG11 mutation is responsible for most of the fluconazole resistance observed in Clade III isolates. We also show that while these mutations likewise affect voriconazole susceptibility, they have no effect on itraconazole, isavuconazole, or posaconazole susceptibility suggesting the therapeutic utility of these antifungals for infections due to Clade III C. auris isolates.