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Stable and reproducible homologous recombination enables CRISPR-based engineering in the fungus Rhizopus microsporus

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dc.contributor.authorLax Molina, Carlos
dc.contributor.authorNavarro Mendoza, María Isabel
dc.contributor.authorPérez Arques, Carlos
dc.contributor.authorNavarro Ros, Eusebio
dc.contributor.authorNicolás Molina, Francisco Esteban
dc.contributor.authorGarre Mula, Victoriano
dc.contributor.departmentGenética y Microbiología
dc.contributor.otherFacultades de la UMU::Facultad de Biología
dc.date.accessioned2026-02-28T22:33:46Z
dc.date.available2026-02-28T22:33:46Z
dc.date.copyright© 2021 The Author(s).
dc.date.issued2021-12-20
dc.description.abstractMucormycosis is a lethal and emerging disease that has lacked a genetic model fulfilling both high virulence and the possibility of performing stable and reproducible gene manipulation by homologous recombination (HR). Here, we developed a new methodology to successfully perform HR in Rhizopus microsporus. We isolated an uracil auxotrophic recipient strain and optimized the critical steps in the genetic transformation of this fungus. This was followed by an adaptation of a plasmid-free CRISPR-Cas9 system coupled with microhomology repair templates. We reproducibly generated stable mutants in the genes leuA and crgA, encoding a 3-isopropylmalate dehydratase and an ubiquitin ligase, respectively. Our new genetic model showed that mutations in the gene pyrF, a key virulence gene in several bacterial and fungal pathogens, correlated with an avirulent phenotype in an immunocompetent murine host. This was reverted by gene complementation, showing the broad possibilities of our methodology.
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dc.format.extent16
dc.identifier.citationLax, C., Navarro-Mendoza, M. I., Pérez-Arques, C., Navarro, E., Nicolás, F. E., & Garre, V. (2021). Stable and reproducible homologous recombination enables CRISPR-based engineering in the fungus Rhizopus microsporus. Cell reports methods, 1(8), 100124. https://doi.org/10.1016/j.crmeth.2021.100124
dc.identifier.doihttps://doi.org/10.1016/j.crmeth.2021.100124
dc.identifier.eissn2667-2375
dc.identifier.urihttp://hdl.handle.net/10201/216521
dc.languageeng
dc.publisherCell Press
dc.relation20897/PI/18. Fundación Séneca. Regional FPU17/05814. MICIU. Nacional
dc.relation.publisherversionhttps://www.sciencedirect.com/science/article/pii/S2667237521001880
dc.rightsAttribution 4.0 International*
dc.rights.accessRightsinfo:eu-repo/semantics/openAccess
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/*
dc.subjectMucormycosis
dc.subjectMucorales
dc.subjectHomologous recombination
dc.subjectGenetic model
dc.subjectRhizopus microsporus
dc.subjectCRISPR
dc.subjectVirulence
dc.subjectpyrF
dc.subjectleuA
dc.subjectcrgA
dc.subject.odsObjetivo 3: Salud
dc.titleStable and reproducible homologous recombination enables CRISPR-based engineering in the fungus Rhizopus microsporus
dc.typeinfo:eu-repo/semantics/article
dc.type.versioninfo:eu-repo/semantics/publishedVersion
dspace.entity.typePublicationes
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