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This highlights the need to develop specific genetic tools to mimic and study genetic amplification in this important fungal pathogen. Here, we report the development, validation, and applications of the first clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) system in\n                      <jats:italic>N. glabrata<\/jats:italic>\n                      for targeted genetic overexpression. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in\n                      <jats:italic>N. glabrata<\/jats:italic>\n                      , and further assess optimal guide RNA targeting for robust overexpression. We demonstrate the applications of CRISPRa to overexpress genes involved in fungal pathogenesis and drug resistance and detect corresponding phenotypic alterations in these key traits, including the characterization of novel phenotypes. Finally, we capture strain variation using our CRISPRa system in two commonly used\n                      <jats:italic>N. glabrata<\/jats:italic>\n                      genetic backgrounds. Together, this tool will expand our capacity for functional genetic overexpression in this pathogen, with numerous possibilities for future applications.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        <jats:italic>Nakaseomyces<\/jats:italic>\n                        (formerly\n                        <jats:italic>Candida<\/jats:italic>\n                        )\n                        <jats:italic>glabrata<\/jats:italic>\n                        is an important fungal pathogen that is now the second leading cause of candidiasis infections. A common strategy that this pathogen employs to resist antifungal treatment is through the upregulation of gene expression, but we have limited tools available to study this phenomenon. Here, we develop, optimize, and apply the use of CRISPRa as a means to overexpress genes in\n                        <jats:italic>N. glabrata<\/jats:italic>\n                        . We demonstrate the utility of this system to overexpress key genes involved in antifungal susceptibility, stress tolerance, and biofilm growth. This tool will be an important contribution to our ability to study the biology of this important fungal pathogen.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/msphere.00761-23","type":"journal-article","created":{"date-parts":[[2024,1,24]],"date-time":"2024-01-24T09:01:16Z","timestamp":1706086876000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":17,"title":["Functional genetic characterization of stress tolerance and biofilm formation in\n                    <i>Nakaseomyces<\/i>\n                    (\n                    <i>Candida<\/i>\n                    )\n                    <i>glabrata<\/i>\n                    via a novel CRISPR activation system"],"prefix":"10.1128","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3144-0511","authenticated-orcid":false,"given":"Laetitia","family":"Maroc","sequence":"first","affiliation":[{"name":"Department of Molecular and Cellular Biology, University of Guelph, Guelph, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hajer","family":"Shaker","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Biology, University of Guelph, Guelph, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7119-8865","authenticated-orcid":false,"given":"Rebecca S.","family":"Shapiro","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Biology, University of Guelph, Guelph, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","unstructured":"WHO fungal priority pathogens list to guide research development and public health action. 2022. 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