{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T20:09:22Z","timestamp":1789762162544,"version":"4.0.1"},"reference-count":51,"publisher":"American Chemical Society (ACS)","issue":"9","license":[{"start":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T00:00:00Z","timestamp":1786492800000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T00:00:00Z","timestamp":1786492800000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T00:00:00Z","timestamp":1786492800000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/501100013290","name":"National Key Research and Development Program of China Stem Cell and Translational Research","doi-asserted-by":"publisher","award":["2022YFC2804203"],"award-info":[{"award-number":["2022YFC2804203"]}],"id":[{"id":"10.13039\/501100013290","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,18]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Fusarium oxysporum, as one of the most common filamentous fungi, possesses great biosynthetic potential for natural products. However, the lack of efficient genetic tools has hindered functional genome mining and metabolic engineering in this fungus. In this study, a novel highly efficient CRISPR\/Cas9-based dual-sgRNA expression editing system for F. oxysporum was successfully developed through construction of a robust plasmid platform pFRCas9-G418 using incorporation of an endogenous histone H2B nuclear localization signal and a 5S rRNA promoter-driven polycistronic tRNA\u2212sgRNA cassette. This system is suitable not only for single-gene editing but also for large-fragment deletion and multiplex gene editing, although the editing efficiency is somewhat lower. First, this new CRISPR\/Cas9 system exhibited a high efficiency of 93.75% \u00b1 6.25% for deletion of the Fusarium cyclin C1 (fcc1) gene (\u223c1 kb), which was usually selected as the target gene responsible for yellow pigment accumulation. Then, knockout of the core NRPS gene sanB (\u223c19 kb) and knock-in of the strong promoter gpdA in the N-methylsansalvamide (SA) biosynthetic gene cluster (BGC) in strain F. oxysporum R1 using this system, respectively, led to no SA yield and an increase of 26.4% SA titer, confirming its capacity for large gene deletion and gene knock-in. Furthermore, one-step dual-gene knockout of hat1 (histone acetyltransferase gene, \u223c1.5 kb) and pacC (pH-responsive transcription factor, \u223c2 kb) was first achieved in Fusarium species. This versatile platform provides a powerful tool for editing gene(s) of various sizes in F. oxysporum.<\/jats:p>","DOI":"10.1021\/acssynbio.6c00299","type":"journal-article","created":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T16:03:55Z","timestamp":1786550635000},"page":"3676-3685","source":"Crossref","is-referenced-by-count":0,"title":["A Novel, Simple, and Highly Efficient Dual-sgRNA CRISPR\/Cas9-Based Editing System in the Filamentous Fungus\n                    <i>Fusarium oxysporum<\/i>"],"prefix":"10.1021","volume":"15","author":[{"given":"Wangjie","family":"Zhu","sequence":"first","affiliation":[{"name":"Zhejiang University of Technology , , ,","place":["Hangzhou, China, 310014"]},{"name":"Zhejiang University of Technology , , ,","place":["Hangzhou, China, 310014"]},{"name":"Zhejiang-Egypt Joint Laboratory on Intelligent Discovery of Marine Drugs , ,","place":["Hangzhou, China, 310014"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiaoqun","family":"Liao","sequence":"additional","affiliation":[{"name":"Zhejiang University of Technology , , ,","place":["Hangzhou, China, 310014"]},{"name":"Zhejiang University of Technology , , ,","place":["Hangzhou, China, 310014"]},{"name":"Zhejiang-Egypt Joint Laboratory on Intelligent Discovery of Marine Drugs , ,","place":["Hangzhou, China, 310014"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuanyuan","family":"Liu","sequence":"additional","affiliation":[{"name":"Zhejiang University of Technology , , ,","place":["Hangzhou, China, 310014"]},{"name":"Zhejiang University of Technology , , ,","place":["Hangzhou, China, 310014"]},{"name":"Zhejiang-Egypt Joint Laboratory on Intelligent Discovery of Marine Drugs , ,","place":["Hangzhou, China, 310014"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2430-3845","authenticated-orcid":true,"given":"Huawei","family":"Zhang","sequence":"additional","affiliation":[{"name":"Zhejiang University of Technology , , ,","place":["Hangzhou, China, 310014"]},{"name":"Zhejiang University of Technology , , ,","place":["Hangzhou, China, 310014"]},{"name":"Zhejiang-Egypt Joint Laboratory on Intelligent Discovery of Marine Drugs , ,","place":["Hangzhou, China, 310014"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,12]]},"reference":[{"key":"2026091815070154000_cit1","doi-asserted-by":"publisher","first-page":"1568","DOI":"10.1039\/d0np00038h","article-title":"Fusarium: a treasure trove of bioactive secondary metabolites","volume":"37","author":"Li","year":"2020","journal-title":"Nat. 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