{"title":"Optimization of the Genome Editing CRISPR-Cas9 Technology in Scedosporium apiospermum.","authors":"Kévin Ravenel, Wilfried Poirier, Bienvenue Razafimandimby, Jean-Philippe Bouchara, Amandine Gastebois, Sandrine Giraud","doi":"10.1007/s11046-025-00998-8","DOIUrl":null,"url":null,"abstract":"<p><p>Scedosporium species are opportunistic pathogens causing a large variety of human infections. To date, there is limited information on the pathogenic mechanisms of these fungi, partly because of the limited number of genetic tools available. Here, the CRISPR-Cas9 technology, which provided promising results for functional genomic studies in filamentous fungi, was optimized for Scedosporium species using in vitro assembled Cas9 ribonucleoprotein (RNP) complexes. In these fungi, functional genomic studies are particularly complex in a wild-type strain, because of the high frequency of non-homologous recombination. Prior disruption of the KU70 gene encoding one of the components of the non-homologous end joining system is required, which necessitates the use of a first selection marker. The cleavage of the target gene at each end using a dual RNA-guided Cas9 complex, followed by recombination with a repair template containing the hygromycin resistance gene, allowed disruption of the target gene in the ΔKU70 mutant. Four genes encoding dioxygenases, catalyzing the critical ring-opening step in aromatic hydrocarbons, were successfully disrupted, and the optimum efficiency was observed using 5 μg of the HygR repair cassette. Alternatively, in the wild-type strain, the exclusive use of two Cas9 RNP complexes was enough to achieve an efficient deletion method; one dioxygenase gene was successfully deleted in up to 20% of the obtained colonies. These last experimental conditions path the way to multiple gene deletions and complementation experiments, which cannot be reached using our first procedure since only two selection markers are available for Scedosporium species.</p>","PeriodicalId":19017,"journal":{"name":"Mycopathologia","volume":"190 6","pages":"94"},"PeriodicalIF":2.9000,"publicationDate":"2025-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Mycopathologia","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1007/s11046-025-00998-8","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"MYCOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Scedosporium species are opportunistic pathogens causing a large variety of human infections. To date, there is limited information on the pathogenic mechanisms of these fungi, partly because of the limited number of genetic tools available. Here, the CRISPR-Cas9 technology, which provided promising results for functional genomic studies in filamentous fungi, was optimized for Scedosporium species using in vitro assembled Cas9 ribonucleoprotein (RNP) complexes. In these fungi, functional genomic studies are particularly complex in a wild-type strain, because of the high frequency of non-homologous recombination. Prior disruption of the KU70 gene encoding one of the components of the non-homologous end joining system is required, which necessitates the use of a first selection marker. The cleavage of the target gene at each end using a dual RNA-guided Cas9 complex, followed by recombination with a repair template containing the hygromycin resistance gene, allowed disruption of the target gene in the ΔKU70 mutant. Four genes encoding dioxygenases, catalyzing the critical ring-opening step in aromatic hydrocarbons, were successfully disrupted, and the optimum efficiency was observed using 5 μg of the HygR repair cassette. Alternatively, in the wild-type strain, the exclusive use of two Cas9 RNP complexes was enough to achieve an efficient deletion method; one dioxygenase gene was successfully deleted in up to 20% of the obtained colonies. These last experimental conditions path the way to multiple gene deletions and complementation experiments, which cannot be reached using our first procedure since only two selection markers are available for Scedosporium species.
期刊介绍:
Mycopathologia is an official journal of the International Union of Microbiological Societies (IUMS). Mycopathologia was founded in 1938 with the mission to ‘diffuse the understanding of fungal diseases in man and animals among mycologists’. Many of the milestones discoveries in the field of medical mycology have been communicated through the pages of this journal. Mycopathologia covers a diverse, interdisciplinary range of topics that is unique in breadth and depth. The journal publishes peer-reviewed, original articles highlighting important developments concerning medically important fungi and fungal diseases. The journal highlights important developments in fungal systematics and taxonomy, laboratory diagnosis of fungal infections, antifungal drugs, clinical presentation and treatment, and epidemiology of fungal diseases globally. Timely opinion articles, mini-reviews, and other communications are usually invited at the discretion of the editorial board. Unique case reports highlighting unprecedented progress in the diagnosis and treatment of fungal infections, are published in every issue of the journal. MycopathologiaIMAGE is another regular feature for a brief clinical report of potential interest to a mixed audience of physicians and laboratory scientists. MycopathologiaGENOME is designed for the rapid publication of new genomes of human and animal pathogenic fungi using a checklist-based, standardized format.