Genome editing in Kluyveromyces and Ogataea yeasts using a broad-host-range Cas9/gRNA co-expression plasmid

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dc.contributor.authorJuergens, Hannes
dc.contributor.authorVarela, Javier A.
dc.contributor.authorGorter de Vries, Arthur R.
dc.contributor.authorPerli, Thomas
dc.contributor.authorGast, Veronica J. M.
dc.contributor.authorGyurchev, Nikola Y.
dc.contributor.authorRajkumar, Arun S.
dc.contributor.authorMans, Robert
dc.contributor.authorPronk, Jack T.
dc.contributor.authorMorrissey, John P.
dc.contributor.authorDaran, Jean-Marc G.
dc.contributor.funderEuropean Research Council
dc.contributor.funderH2020 Marie Skłodowska-Curie Actions
dc.contributor.funderHorizon 2020 Framework Programme
dc.contributor.funderMinistry of Economic Affairs, Agriculture and Innovation
dc.date.accessioned2018-09-27T12:08:22Z
dc.date.available2018-09-27T12:08:22Z
dc.date.issued2018
dc.description.abstractWhile CRISPR-Cas9-mediated genome editing has transformed yeast research, current plasmids and cassettes for Cas9 and guide-RNA expression are species specific. CRISPR tools that function in multiple yeast species could contribute to the intensifying research on non-conventional yeasts. A plasmid carrying a pangenomic origin of replication and two constitutive expression cassettes for Cas9 and ribozyme-flanked gRNAs was constructed. Its functionality was tested by analyzing inactivation of the ADE2 gene in four yeast species. In two Kluyveromyces species, near-perfect targeting (≥96%) and homologous repair (HR) were observed in at least 24% of transformants. In two Ogataea species, Ade− mutants were not observed directly after transformation, but prolonged incubation of transformed cells resulted in targeting efficiencies of 9% to 63% mediated by non-homologous end joining (NHEJ). In an Ogataea parapolymorpha ku80 mutant, deletion of OpADE2 mediated by HR was achieved, albeit at low efficiencies (<1%). Furthermore the expression of a dual polycistronic gRNA array enabled simultaneous interruption of OpADE2 and OpYNR1 demonstrating flexibility of ribozyme-flanked gRNA design for multiplexing. While prevalence of NHEJ prevented HR-mediated editing in Ogataea, such targeted editing was possible in Kluyveromyces. This broad-host-range CRISPR/gRNA system may contribute to exploration of Cas9-mediated genome editing in other Saccharomycotina yeastsen
dc.description.sponsorshipMinistry of Economic Affairs, Agriculture and Innovation (BE-Basic R&D Program).en
dc.description.statusPeer revieweden
dc.description.versionPublished Versionen
dc.format.mimetypeapplication/pdfen
dc.identifier.articleidfoy012
dc.identifier.citationJuergens, H., Varela, J. A., Gorter de Vries, A. R., Perli, T., Gast, V. J. M., Gyurchev, N. Y., Rajkumar, A. S., Mans, R., Pronk, J. T., Morrissey, J. P. and Daran, J.-M. G. (2018) 'Genome editing in Kluyveromyces and Ogataea yeasts using a broad-host-range Cas9/gRNA co-expression plasmid', FEMS Yeast Research, 18(3), foy012(16pp). doi: 10.1093/femsyr/foy012en
dc.identifier.doi10.1093/femsyr/foy012
dc.identifier.endpage18
dc.identifier.issn1567-1356
dc.identifier.issn1567-1364
dc.identifier.issued3
dc.identifier.journaltitleFEMS Yeast Researchen
dc.identifier.startpage1
dc.identifier.urihttps://hdl.handle.net/10468/6941
dc.identifier.volume18
dc.language.isoenen
dc.publisherOxford University Pressen
dc.relation.projectinfo:eu-repo/grantAgreement/EC/FP7::SP3::PEOPLE/606795/EU/Yeast Cell Factories: Training Researchers to Apply Modern Post-Genomic Methods In Yeast Biotechnology/YEASTCELL
dc.relation.projectinfo:eu-repo/grantAgreement/EC/H2020::RIA/720824/EU/Model-Based Construction And Optimisation Of Versatile Chassis Yeast Strains For Production Of Valuable Lipid And Aromatic Compounds/CHASSY
dc.relation.projectinfo:eu-repo/grantAgreement/EC/H2020::ERC::ERC-ADG/694633/EU/Eliminating Oxygen Requirements in Yeasts/ELOXY
dc.relation.projectinfo:eu-repo/grantAgreement/EC/H2020::MSCA-ITN-ETN/722287/EU/Predictive and Accelerated Metabolic Engineering Network/PAcMEN
dc.relation.urihttps://academic.oup.com/femsyr/article/18/3/foy012/4847887
dc.rights© 2018, FEMS. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.comen
dc.rights.urihttp://creativecommons.org/licenses/by-nc/4.0/
dc.subjectCrispr/cas9en
dc.subjectRibozymesen
dc.subjectKluyveromyces lactisen
dc.subjectKluyveromyces marxianusen
dc.subjectOgataea polymorphaen
dc.subjectHansenula polymorphaen
dc.titleGenome editing in Kluyveromyces and Ogataea yeasts using a broad-host-range Cas9/gRNA co-expression plasmiden
dc.typeArticle (peer-reviewed)en
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