A pan-azole-resistant strain with the mutations Gly138Ser and Asn248Lys was isolated from a patient receiving long-term voriconazole treatment. PCR fragments containing with the mutations were introduced along with the Cas9 protein and single guide RNA into the azole-resistant/susceptible strains. Recombinant strains showed increased susceptibility via the replacement of Ser138 by glycine. Genetic recombination, which has been hampered thus far in clinical isolates, can now be achieved using CRISPR/Cas9 genome editing.

23Azole resistance in Aspergillus fumigatus is predominantly associated with increased expression 24 of Cyp51A (lanosterol 14α-demethylase), the target enzyme of azole antifungal agents, or with 25 single-nucleotide polymorphisms (SNPs) in cyp51A. Although several SNPs that may be linked 26 to low susceptibility in azole-resistant isolates have previously been reported, few studies have 27 been conducted to conclusively demonstrate the contribution of SNPs to decreased azole 28susceptibility. An A. fumigatus strain was isolated from the sputum of a 74-year-old male 29 receiving long-term voriconazole treatment for chronic progressive pulmonary aspergillosis. 30Etest antifungal susceptibility testing showed low susceptibility to voriconazole, itraconazole, 31 and posaconazole. Nucleotide sequencing of cyp51A from this isolate revealed the mutations 32 Gly138Ser (GGC→AGC) and Asn248Lys (AAT AAA) compared with the cyp51A of 33 azole-susceptible isolates. PCR-amplified DNA fragments containing cyp51A with or without the 34 mutations of interest and a hygromycin marker were simultaneously introduced along with the 35 Cas9 protein and in vitro-synthesized single-guide RNA into protoplasts of the 36 azole-resistant/susceptible strains. Etest azole susceptibility testing of recombinant strains 37 showed an increased susceptibility via the replacement of Ser138 by glycine. In contrast, azole 38 susceptibility was slightly decreased when a Ser138 mutation was introduced into the 39 azole-susceptible strain AfS35, indicating that the serine at position 138 of Cyp51A contributes 40 to low susceptibility in the azole-resistant isolate. Genetic recombination, which has been 41 hampered thus far in clinical isolates, can now be achieved using Cas9/CRISPR genome editing. 42This technique could be useful to investigate the contribution of other SNPs of cyp51A to azole 43 resistance. 44 45