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However, prior studies in these pathogens have been mainly limited to the use of two drug resistance cassettes,\n                    <jats:italic>NatMX<\/jats:italic>\n                    and\n                    <jats:italic>HphMX<\/jats:italic>\n                    , limiting genetic manipulation capabilities in prototrophic laboratory strains and clinical isolates. In this study, we expanded the toolkit for\n                    <jats:italic>C. glabrata<\/jats:italic>\n                    ,\n                    <jats:italic>C. auris<\/jats:italic>\n                    , and\n                    <jats:italic>C. albicans<\/jats:italic>\n                    to include\n                    <jats:italic>KanMX<\/jats:italic>\n                    and\n                    <jats:italic>BleMX<\/jats:italic>\n                    when coupled with an\n                    <jats:italic>in vitro<\/jats:italic>\n                    assembled CRISPR-Cas9 ribonucleoprotein (RNP)-based system. Repurposing these drug resistance cassettes for\n                    <jats:italic>Candida<\/jats:italic>\n                    , we were able to make single gene deletions, sequential and simultaneous double gene deletions, epitope tags, and rescue constructs. We applied these drug resistance cassettes to interrogate the ergosterol pathway, a critical pathway for both the azole and polyene antifungal drug classes. Using our approach, we determined for the first time that the deletion of\n                    <jats:italic>ERG3<\/jats:italic>\n                    in\n                    <jats:italic>C. glabrata<\/jats:italic>\n                    ,\n                    <jats:italic>C. auris,<\/jats:italic>\n                    and\n                    <jats:italic>C. albicans<\/jats:italic>\n                    prototrophic strains results in azole drug resistance, which further supports the conservation of the Erg3-dependent toxic sterol model. Furthermore, we show that an\n                    <jats:italic>ERG5<\/jats:italic>\n                    deletion in\n                    <jats:italic>C. glabrata<\/jats:italic>\n                    is azole susceptible at subinhibitory concentrations, suggesting that Erg5 could act as an azole buffer for Erg11. Finally, we identified a synthetic growth defect when both\n                    <jats:italic>ERG3<\/jats:italic>\n                    and\n                    <jats:italic>ERG5<\/jats:italic>\n                    are deleted in\n                    <jats:italic>C. glabrata,<\/jats:italic>\n                    which suggests the possibility of another toxic sterol impacting growth. Overall, we have expanded the genetic tools available to interrogate complex pathways in prototrophic strains and clinical isolates.\n                  <\/jats:p>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      The increasing problem of drug resistance and emerging pathogens is an urgent global health problem that necessitates the development and expansion of tools for studying fungal drug resistance and pathogenesis. Prior studies in\n                      <jats:italic>Candida glabrata<\/jats:italic>\n                      ,\n                      <jats:italic>Candida auris<\/jats:italic>\n                      , and\n                      <jats:italic>Candida albicans<\/jats:italic>\n                      have been mainly limited to the use of\n                      <jats:italic>NatMX\/SAT1<\/jats:italic>\n                      and\n                      <jats:italic>HphMX\/CaHyg<\/jats:italic>\n                      for genetic manipulation in prototrophic strains and clinical isolates. In this study, we demonstrated that\n                      <jats:italic>NatMX\/SAT1, HphMX, KanMX,<\/jats:italic>\n                      and\/or\n                      <jats:italic>BleMX<\/jats:italic>\n                      drug resistance cassettes when coupled with a CRISPR-ribonucleoprotein (RNP)-based system can be efficiently utilized for deleting or modifying genes in the ergosterol pathway of\n                      <jats:italic>C. glabrata<\/jats:italic>\n                      ,\n                      <jats:italic>C. auris<\/jats:italic>\n                      , and\n                      <jats:italic>C. albicans<\/jats:italic>\n                      . Moreover, the utility of these tools has provided new insights into\n                      <jats:italic>ERG<\/jats:italic>\n                      genes and their relationship to azole resistance in\n                      <jats:italic>Candida<\/jats:italic>\n                      . Overall, we have expanded the toolkit for\n                      <jats:italic>Candida<\/jats:italic>\n                      pathogens to increase the versatility of genetically modifying complex pathways involved in drug resistance and pathogenesis.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/msphere.00311-23","type":"journal-article","created":{"date-parts":[[2023,11,6]],"date-time":"2023-11-06T08:00:27Z","timestamp":1699257627000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":20,"title":["An expanded toolkit of drug resistance cassettes for\n                    <i>Candida glabrata<\/i>\n                    ,\n                    <i>Candida auris<\/i>\n                    , and\n                    <i>Candida albicans<\/i>\n                    leads to new insights into the ergosterol pathway"],"prefix":"10.1128","volume":"8","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2634-3178","authenticated-orcid":false,"given":"Justin B.","family":"Gregor","sequence":"first","affiliation":[{"name":"Department of Biochemistry, Purdue University, West Lafayette, Indiana, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Victor A.","family":"Gutierrez-Schultz","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Purdue University, West Lafayette, Indiana, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Smriti","family":"Hoda","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Purdue University, West Lafayette, Indiana, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kortany M.","family":"Baker","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Purdue University, West Lafayette, Indiana, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Debasmita","family":"Saha","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Purdue University, West Lafayette, Indiana, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Madeline G.","family":"Burghaze","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Purdue University, West Lafayette, Indiana, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cynthia","family":"Vazquez","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Purdue University, West Lafayette, Indiana, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kendra E.","family":"Burgei","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Purdue University, West Lafayette, Indiana, 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