Strategies for genetic manipulation of the halotolerant black yeast Hortaea werneckii: ectopic DNA integration and marker-free CRISPR/Cas9 transformation

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dc.contributor.authorHernandez-Rodriguez, Yainitzaen
dc.contributor.authorBullard, A. Makenzieen
dc.contributor.authorBusch, Rebecca J.en
dc.contributor.authorMarshall, Aidanen
dc.contributor.authorVargas-Muñiz, José M.en
dc.date.accessioned2025-01-22T14:07:28Zen
dc.date.available2025-01-22T14:07:28Zen
dc.date.issued2024-12-10en
dc.description.abstractHortaea werneckii is a halotolerant black yeast commonly found in hypersaline environments. This yeast is also the causative agent of tinea nigra, a superficial mycosis of the palm of the hand and soles of the feet of humans. In addition to their remarkable halotolerance, this black yeast exhibits an unconventional cell division cycle, alternating between fission and budding cell division. Cell density and the salt concentration in their environment regulate which cell division cycle H. werneckii uses. Although H. werneckii have been extensively studied due to their unique physiology and cell biology, deciphering the underlying mechanisms behind these remarkable phenotypes has been limited due to the lack of genetic tools available. Here, we report a new ectopic integration protocol for H. werneckii using polyethylene glycol-CaCl2 mediated protoplast transformation. This approach relies on a drug (hygromycin B) resistance gene to select for successful integration of the genetic construct. The same construct was used to express cytosolic green fluorescent protein. Finally, we developed a marker-free CRISPR/Cas9 protocol for targeted gene deletion using the melanin synthesis pathway as a visual reporter of successful transformation. These transformation strategies will allow testing hypotheses related to H. werneckii cell biology and physiology. IMPORTANCE Hortaea werneckii is a remarkable yeast capable of growing in high salt concentration, and its cell division cycle alternates between fission-like and budding. For these unique attributes, H. werneckii has gathered interest in research programs studying extremophile fungi and cell division. Most of our understanding of H. werneckii biology comes from genomic analyses, the usage of drugs to target a particular pathway, or the heterologous expression of its genes in S. cerevisiae. Nonetheless, H. werneckii has remained genetically intractable. Here, we report on two strategies to transform H. werneckii: ectopic integration of a plasmid and gene deletion using CRISPR/Cas9. These approaches will be fundamental to expanding the experimental techniques available to study H. werneckii, including live-cell imaging of cellular processes and reverse genetic approaches.en
dc.description.versionPublished versionen
dc.format.extent10 page(s)en
dc.format.mimetypeapplication/pdfen
dc.identifier.doihttps://doi.org/10.1128/spectrum.02430-24en
dc.identifier.eissn2165-0497en
dc.identifier.issn2165-0497en
dc.identifier.issue1en
dc.identifier.orcidVargas-Muniz, Jose [0000-0003-0138-8660]en
dc.identifier.pmid39656015en
dc.identifier.urihttps://hdl.handle.net/10919/124305en
dc.identifier.volume13en
dc.language.isoenen
dc.publisherAmerican Society for Microbiologyen
dc.relation.urihttps://www.ncbi.nlm.nih.gov/pubmed/39656015en
dc.rightsCreative Commons Attribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.subjectHortaea werneckiien
dc.subjecttransformationen
dc.subjectmorphologyen
dc.subjectectopic integrationen
dc.subject.meshCRISPR-Cas Systemsen
dc.subject.meshTransformation, Geneticen
dc.subject.meshGene Editingen
dc.subject.meshAscomycotaen
dc.subject.meshDNA, Fungalen
dc.titleStrategies for genetic manipulation of the halotolerant black yeast <i>Hortaea werneckii</i>: ectopic DNA integration and marker-free CRISPR/Cas9 transformationen
dc.title.serialMicrobiology Spectrumen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten
dc.type.otherArticleen
dc.type.otherJournalen
pubs.organisational-groupVirginia Techen
pubs.organisational-groupVirginia Tech/Scienceen
pubs.organisational-groupVirginia Tech/Science/Biological Sciencesen
pubs.organisational-groupVirginia Tech/All T&R Facultyen
pubs.organisational-groupVirginia Tech/Science/COS T&R Facultyen

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