{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,13]],"date-time":"2026-10-13T22:33:48Z","timestamp":1791930828772,"version":"4.3.4"},"reference-count":23,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2019,8,7]],"date-time":"2019-08-07T00:00:00Z","timestamp":1565136000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100004440","name":"Wellcome Trust","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100004440","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100008831","name":"Leibniz-Institut f\u00fcr Naturstoff-Forschung und Infektionsbiologie \u2013 Hans-Kn\u00f6ll-Institut","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100008831","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["currentprotocols.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["CP Microbiology"],"published-print":{"date-parts":[[2019,9]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p><jats:italic>Aspergillus fumigatus<\/jats:italic> is a human pathogen and the principal etiologic agent of invasive and chronic aspergillosis leading to several hundreds of thousands of deaths every year. Very few antifungals are available to treat infections caused by <jats:italic>A. fumigatus<\/jats:italic>, and resistance is developing to those we have. Our understanding of the molecular mechanisms that drive pathogenicity and drug resistance have been hampered by the lack of large mutant collections, which limits our ability to perform functional genomics analysis. Here we present a high\u2010throughput gene knockout method that combines a highly reproducible fusion PCR method to enable generation of gene replacement cassettes with a multiwell format transformation procedure. This process can be used to generate 96 null mutants within 5 days by a single person at a cost of less than \u00a318 ($24) per mutant and is being employed in our laboratory to generate a barcoded genome\u2010wide knockout library in <jats:italic>A. fumigatus<\/jats:italic>. \u00a9 2019 The Authors.<\/jats:p>","DOI":"10.1002\/cpmc.88","type":"journal-article","created":{"date-parts":[[2019,9,1]],"date-time":"2019-09-01T15:37:27Z","timestamp":1567352247000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":68,"title":["High\u2010Throughput Gene Replacement in <i>Aspergillus fumigatus<\/i>"],"prefix":"10.1002","volume":"54","author":[{"given":"Can","family":"Zhao","sequence":"first","affiliation":[{"name":"Manchester Fungal Infection Group, Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, Core Technology Facility University of Manchester  Manchester United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marcin G.","family":"Fraczek","sequence":"additional","affiliation":[{"name":"Division of Evolution &amp; Genomic Sciences, School of Biological Sciences, Faculty of Biology Medicine and Health, Manchester Institute of Biotechnology University of Manchester  Manchester United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lauren","family":"Dineen","sequence":"additional","affiliation":[{"name":"Manchester Fungal Infection Group, Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, Core Technology Facility University of Manchester  Manchester United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ressa","family":"Lebedinec","sequence":"additional","affiliation":[{"name":"Manchester Fungal Infection Group, Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, Core Technology Facility University of Manchester  Manchester United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juliane","family":"Macheleidt","sequence":"additional","affiliation":[{"name":"Department of Molecular and Applied Microbiology Leibniz Institute for Natural Product Research and Infection Biology, Hans Kn\u00f6ll Institute  Jena Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thorsten","family":"Heinekamp","sequence":"additional","affiliation":[{"name":"Department of Molecular and Applied Microbiology Leibniz Institute for Natural Product Research and Infection Biology, Hans Kn\u00f6ll Institute  Jena Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniela","family":"Delneri","sequence":"additional","affiliation":[{"name":"Division of Evolution &amp; Genomic Sciences, School of Biological Sciences, Faculty of Biology Medicine and Health, Manchester Institute of Biotechnology University of Manchester  Manchester United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paul","family":"Bowyer","sequence":"additional","affiliation":[{"name":"Manchester Fungal Infection Group, Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, Core Technology Facility University of Manchester  Manchester United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Axel A.","family":"Brakhage","sequence":"additional","affiliation":[{"name":"Department of Molecular and Applied Microbiology Leibniz Institute for Natural Product Research and Infection Biology, Hans Kn\u00f6ll Institute  Jena Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Bromley","sequence":"additional","affiliation":[{"name":"Manchester Fungal Infection Group, Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, Core Technology Facility University of Manchester  Manchester United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2019,8,7]]},"reference":[{"key":"e_1_2_7_2_1_1","doi-asserted-by":"publisher","DOI":"10.3390\/jof3040057"},{"key":"e_1_2_7_2_2_1","first-page":"165rv13","article-title":"Hidden killers: Human fungal infections","volume":"165","author":"Brown G. 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See above."},{"key":"e_1_2_7_4_2_1","unstructured":"http:\/\/www.gaffi.org"},{"key":"e_1_2_7_4_4_1","unstructured":"http:\/\/www.aspgd.org"},{"key":"e_1_2_7_4_6_1","unstructured":"https:\/\/probes.pw.usda.gov\/batchprimer3"},{"key":"e_1_2_7_4_8_1","unstructured":"https:\/\/fungi.ensembl.org\/biomart\/martview"},{"key":"e_1_2_7_4_10_1","unstructured":"https:\/\/www.rstudio.com\/products\/rstudio\/download"},{"key":"e_1_2_7_4_12_1","unstructured":"https:\/\/gist.github.com\/laurendineen\/89d77c0401c917b71c0287e80a7b935a"}],"container-title":["Current Protocols in Microbiology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpmc.88","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/cpmc.88","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpmc.88","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,9,7]],"date-time":"2023-09-07T21:48:29Z","timestamp":1694123309000},"score":1,"resource":{"primary":{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/10.1002\/cpmc.88"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,8,7]]},"references-count":23,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2019,9]]}},"alternative-id":["10.1002\/cpmc.88"],"URL":"https:\/\/doi.org\/10.1002\/cpmc.88","archive":["Portico"],"relation":{},"ISSN":["1934-8525","1934-8533"],"issn-type":[{"value":"1934-8525","type":"print"},{"value":"1934-8533","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,8,7]]},"assertion":[{"value":"2019-08-07","order":2,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e88"}}