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Tools for Knufia petricola: new techniques for CRISPR/Cas9-based genome editing

  • Black microcolonial fungi represent a group of ascomycetes with similar adaptations for existing in natural and anthropogenically created extreme habitats. They exhibit slow yeast-like or meristematic growth, do not form specialized reproduction structures and accumulate the black pigment 1,8-dihydroxynaphthalene (DHN) in the multilayered cell walls. We chose the rock inhabitant Knufia petricola of the Chaetothyriales as a representative for developing methods for genetic manipulation, simulation of mineral weathering and study of symbiotic interactions. Here, we report on the expansion of the genetic toolkit by more efficient multiplex CRISPR/Cas9 using a plasmid-based system for expression of Cas9 and multiple sgRNAs and three additional resistance selection markers. The targeted integration of expression constructs by replacement of essential genes for pigment synthesis allows for an additional color screening of the transformants. The black-pink screening due to the elimination ofBlack microcolonial fungi represent a group of ascomycetes with similar adaptations for existing in natural and anthropogenically created extreme habitats. They exhibit slow yeast-like or meristematic growth, do not form specialized reproduction structures and accumulate the black pigment 1,8-dihydroxynaphthalene (DHN) in the multilayered cell walls. We chose the rock inhabitant Knufia petricola of the Chaetothyriales as a representative for developing methods for genetic manipulation, simulation of mineral weathering and study of symbiotic interactions. Here, we report on the expansion of the genetic toolkit by more efficient multiplex CRISPR/Cas9 using a plasmid-based system for expression of Cas9 and multiple sgRNAs and three additional resistance selection markers. The targeted integration of expression constructs by replacement of essential genes for pigment synthesis allows for an additional color screening of the transformants. The black-pink screening due to the elimination of pks1 (melanin) was applied for promoter studies using GFP fluorescence as reporter, while the black-white screening due to the concurrent elimination of pks1 (melanin) and phs1 (carotenoids) was used to identify transformants that contain the two expression constructs for co-localization or bimolecular fluorescence complementation (BiFC) studies. In addition, two intergenic regions (igr1, igr2) were identified in which expression constructs can be inserted without causing obvious phenotypes. Plasmids of the pNXR-XXX series (Schumacher, 2012) and new compatible entry plasmids were used for fast and easy generation of expression constructs and are suitable for use in other fungal systems as well.zeige mehrzeige weniger

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Metadaten
Autor*innen:Eileen Erdmann
Koautor*innen:Sarah Nitsche, Anna Gorbushina, Julia Schumacher
Dokumenttyp:Posterpräsentation
Veröffentlichungsform:Präsentation
Sprache:Englisch
Jahr der Erstveröffentlichung:2022
Organisationseinheit der BAM:4 Material und Umwelt
4 Material und Umwelt / 4.0 Abteilungsleitung und andere
DDC-Klassifikation:Technik, Medizin, angewandte Wissenschaften / Ingenieurwissenschaften / Sanitär- und Kommunaltechnik; Umwelttechnik
Freie Schlagwörter:Fluorescent proteins; Genetic engineering; Microcolonial fungi
Themenfelder/Aktivitätsfelder der BAM:Umwelt
Umwelt / Umwelt-Material-Interaktionen
Veranstaltung:31st Fungal Genetics Conference
Veranstaltungsort:USA, CA, Pacific Grove
Beginndatum der Veranstaltung:15.03.2022
Enddatum der Veranstaltung:20.03.2022
Verfügbarkeit des Dokuments:Datei im Netzwerk der BAM verfügbar ("Closed Access")
Datum der Freischaltung:04.04.2022
Referierte Publikation:Nein
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