{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T23:56:59Z","timestamp":1768521419007,"version":"3.49.0"},"posted":{"date-parts":[[2025,2,15]]},"group-title":"Microbiology","reference-count":23,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,2,15]],"date-time":"2025-02-15T00:00:00Z","timestamp":1739577600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,2,15]]},"abstract":"<jats:p>\n                  <jats:bold>Objectives<\/jats:bold>\n                  : Mutations in the\n                  <jats:italic>Candidozyma (Candida) auris<\/jats:italic>\n                  \u03b2-glucan synthase gene\n                  <jats:italic>(FKS1)<\/jats:italic>\n                  altering S639 are frequently associated with clinical echinocandin resistance. However, the direct impact of these mutations remains uncharacterized. We have developed a novel\n                  <jats:italic>C. auris<\/jats:italic>\n                  -optimized Episomal Plasmid-Induced Cas9 (EPIC) gene-editing system capable of recyclable precision genome editing and demonstrate the contribution of\n                  <jats:italic>FKS1<\/jats:italic>\n                  <jats:sup>S639F<\/jats:sup>\n                  mutation to echinocandin resistance.\n                  <jats:bold>Methods<\/jats:bold>\n                  : The EPIC gene-editing system was generated for optimized use in\n                  <jats:italic>C. auris<\/jats:italic>\n                  , and\n                  <jats:italic>ADE2<\/jats:italic>\n                  modification was evaluated in 5\n                  <jats:italic>C. auris<\/jats:italic>\n                  clades. Mutations leading to Fks1\n                  <jats:sup>S639F<\/jats:sup>\n                  into echinocandin-susceptible and echinocandin-resistant isolates from Clade-III and -I, respectively, using the EPIC system. Echinocandin susceptibility was determined by CLSI broth microdilution, and cell wall abundance of chitin and \u03b2-glucan was assessed by staining with Calcofluor White (CFW) and Aniline Blue (AB).\n                  <jats:bold>Results<\/jats:bold>\n                  : The EPIC system was capable of targeted\n                  <jats:italic>ADE2<\/jats:italic>\n                  editing in\n                  <jats:italic>C. auris<\/jats:italic>\n                  isolates from 5 genetic clades and shown to be precise by confirmatory sequencing of 50 transformants. A single nucleotide change in\n                  <jats:italic>FKS1<\/jats:italic>\n                  resulting in either the S639F substitution or a silent synonymous mutation was introduced in an echinocandin-susceptible Clade-III isolate. Precision\n                  <jats:italic>FKS1<\/jats:italic>\n                  editing by the EPIC system was confirmed by whole genome sequencing. Subsequent susceptibility testing demonstrated introduction of the\n                  <jats:italic>FKS1<\/jats:italic>\n                  S639F mutation to increase resistance to echinocandins. Moreover, introduction of a wildtype Fks1 sequence to an echinocandin-resistant Clade-I clinical isolate, correcting the\n                  <jats:italic>FKS1<\/jats:italic>\n                  <jats:sup>S639F<\/jats:sup>\n                  mutation, resulted in a restoration of echinocandin sensitivity. Evaluation of cell wall composition showed isolates or strains harboring\n                  <jats:italic>FKS1<\/jats:italic>\n                  <jats:sup>S639F<\/jats:sup>\n                  to contain significantly elevated \u03b2-glucan and chitin content relative to isogenic comparators.\n                  <jats:bold>Conclusions<\/jats:bold>\n                  : These data demonstrate the potential of our EPIC system in its ability to introduce single nucleotide substitutions in multiple\n                  <jats:italic>C. auris<\/jats:italic>\n                  clade backgrounds while revealing the direct impact of the S639F encoding\n                  <jats:italic>FKS1<\/jats:italic>\n                  mutation on echinocandin resistance.\n                <\/jats:p>","DOI":"10.1101\/2025.02.14.638356","type":"posted-content","created":{"date-parts":[[2025,2,15]],"date-time":"2025-02-15T22:25:21Z","timestamp":1739658321000},"source":"Crossref","is-referenced-by-count":2,"title":["A\n                  <i>Candidozyma (Candida) auris<\/i>\n                  -optimized Episomal Plasmid Induced Cas9-editing system reveals the direct impact of the S639F encoding\n                  <i>FKS1<\/i>\n                  mutation"],"prefix":"10.64898","author":[{"given":"Laura A","family":"Doorley","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vanessa","family":"Meza-Perez","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sarah J","family":"Jones","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jeffrey M","family":"Rybak","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2025021804201950000_2025.02.14.638356v1.1","unstructured":"Centers for Disease Control and Prevention. Antibiotic Resistance Threats in the United States. Atlanta, GA: U.S. Department of Health and Human Services, CDC; 2019."},{"key":"2025021804201950000_2025.02.14.638356v1.2","doi-asserted-by":"publisher","DOI":"10.1093\/cid\/ciw691"},{"key":"2025021804201950000_2025.02.14.638356v1.3","unstructured":"Centers for Disease Control and Prevention. Antifungal Susceptibility Testing for C. auris [updated April 23, 2024. Available from: https:\/\/www.cdc.gov\/candida-auris\/hcp\/laboratories\/antifungal-susceptibility-testing.html."},{"key":"2025021804201950000_2025.02.14.638356v1.4","doi-asserted-by":"publisher","DOI":"10.1111\/myc.13655"},{"key":"2025021804201950000_2025.02.14.638356v1.5","doi-asserted-by":"publisher","DOI":"10.1093\/ofid\/ofz262"},{"key":"2025021804201950000_2025.02.14.638356v1.6","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.00349-06"},{"key":"2025021804201950000_2025.02.14.638356v1.7","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.49.8.3264-3273.2005"},{"key":"2025021804201950000_2025.02.14.638356v1.8","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01653-05"},{"key":"2025021804201950000_2025.02.14.638356v1.9","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.00027-12"},{"key":"2025021804201950000_2025.02.14.638356v1.10","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.02367-14"},{"key":"2025021804201950000_2025.02.14.638356v1.11","doi-asserted-by":"publisher","DOI":"10.1093\/cid\/ciu407"},{"key":"2025021804201950000_2025.02.14.638356v1.12","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01811-10"},{"key":"2025021804201950000_2025.02.14.638356v1.13","doi-asserted-by":"publisher","DOI":"10.1128\/EC.00082-12"},{"key":"2025021804201950000_2025.02.14.638356v1.14","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-023-05856-5"},{"key":"2025021804201950000_2025.02.14.638356v1.15","doi-asserted-by":"publisher","DOI":"10.1080\/22221751.2024.2377584"},{"issue":"6","key":"2025021804201950000_2025.02.14.638356v1.16","first-page":"e00423","article-title":"Novel Non-Hot Spot Modification in Fks1 of Candida auris Confers Echinocandin Resistance","volume":"67","year":"2023","journal-title":"Antimicrobial Agents and Chemotherapy"},{"key":"2025021804201950000_2025.02.14.638356v1.17","doi-asserted-by":"publisher","DOI":"10.3390\/jof9050529"},{"key":"2025021804201950000_2025.02.14.638356v1.18","doi-asserted-by":"publisher","DOI":"10.1016\/j.cmi.2021.11.024"},{"key":"2025021804201950000_2025.02.14.638356v1.19","doi-asserted-by":"publisher","DOI":"10.1128\/mSphere.00125-19"},{"key":"2025021804201950000_2025.02.14.638356v1.20","unstructured":"Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087 USA: Clinical and Laboratory Standards Institute; 2017. ISBN: 1-56238-826-6"},{"key":"2025021804201950000_2025.02.14.638356v1.21","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkx480"},{"key":"2025021804201950000_2025.02.14.638356v1.22","doi-asserted-by":"publisher","DOI":"10.1093\/infdis\/jir351"},{"key":"2025021804201950000_2025.02.14.638356v1.23","unstructured":"Clinical and Laboratory Standards Institute. Performance standard for Antifungal Susceptibility Testing of Yeasts. USA: Clinical and Laboratory Standards Institute; 2022. ISBN: 978-1-68440-163-5"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.02.14.638356","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T08:03:33Z","timestamp":1768464213000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.02.14.638356"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,2,15]]},"references-count":23,"URL":"https:\/\/doi.org\/10.1101\/2025.02.14.638356","relation":{"is-preprint-of":[{"id-type":"doi","id":"10.1093\/infdis\/jiaf285","asserted-by":"subject"}]},"subject":[],"published":{"date-parts":[[2025,2,15]]},"subtype":"preprint"}}