Azole resistance inAspergillus fumigatusis predominantly associated with increased expression of Cyp51A (lanosterol 14α-demethylase), the target enzyme of azole antifungal agents, or with single-nucleotide polymorphisms (SNPs) incyp51A. Although several SNPs that may be linked to low susceptibility in azole-resistant isolates have previously been reported, few studies have been conducted to conclusively demonstrate the contribution of SNPs to decreased azole susceptibility. AnA. fumigatusstrain

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... was isolated from the sputum of a 74-year-old male receiving long-term voriconazole treatment for chronic progressive pulmonary aspergillosis. Etest antifungal susceptibility testing showed low susceptibility to voriconazole, itraconazole, and posaconazole. Nucleotide sequencing ofcyp51Afrom this isolate revealed the mutations Gly138Ser (GGC→AGC) and Asn248Lys (AAT→AAA) compared with thecyp51Aof azole-susceptible isolates. PCR-amplified DNA fragments containingcyp51Awith or without the mutations of interest and a hygromycin marker were simultaneously introduced along with the Cas9 protein andin vitro-synthesized single-guide RNA into protoplasts of the azole-resistant/susceptible strains. Etest azole susceptibility testing of recombinant strains showed an increased susceptibility via the replacement of Ser138 by glycine. In contrast, azole susceptibility was slightly decreased when a Ser138 mutation was introduced into the azole-susceptible strain AfS35, indicating that the serine at position 138 of Cyp51A contributes to low susceptibility in the azole-resistant isolate. Genetic recombination, which has been hampered thus far in clinical isolates, can now be achieved using Cas9/CRISPR genome editing. This technique could be useful to investigate the contribution of other SNPs ofcyp51Ato azole resistance.