RT Journal Article SR Electronic T1 Cas9/CRISPR genome editing to demonstrate the contribution of Cyp51A Gly138Ser to azole resistance in Aspergillus fumigatus JF bioRxiv FD Cold Spring Harbor Laboratory SP 311712 DO 10.1101/311712 A1 Takashi Umeyama A1 Yuta Hayashi A1 Hisaki Shimosaka A1 Tatsuya Inukai A1 Satoshi Yamagoe A1 Shogo Takatsuka A1 Yasutaka Hoshino A1 Minoru Nagi A1 Shigeki Nakamura A1 Katsuhiko Kamei A1 Kenji Ogawa A1 Yoshitsugu Miyazaki YR 2018 UL http://biorxiv.org/content/early/2018/05/01/311712.abstract AB Azole resistance in Aspergillus fumigatus is predominantly associated with increased expression of Cyp51A (lanosterol 14α-demethylase), the target enzyme of azole antifungal agents, or with single-nucleotide polymorphisms (SNPs) in cyp51A. 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. An A. fumigatus strain 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 of cyp51A from this isolate revealed the mutations Gly138Ser (GGC→AGC) and Asn248Lys (AAT→AAA) compared with the cyp51A of azole-susceptible isolates. PCR-amplified DNA fragments containing cyp51A with or without the mutations of interest and a hygromycin marker were simultaneously introduced along with the Cas9 protein and in 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 of cyp51A to azole resistance.