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|Title:||Genome editing in Kluyveromyces and Ogataea yeasts using a broad-host-range Cas9/gRNA co-expression plasmid.|
Varela, Javier A.
Gorter de Vries, Arthur R.
Gast, Veronica J. M.
Gyurchev, Nikola Y.
Rajkumar, Arun S.
Pronk, Jack T.
Morrissey, John P.
Daran, Jean-Mark G.
|Publisher:||Oxford University Press (OUP) for Federation of European Microbiological Societies|
|Citation:||FEMS Yeast Res, 2018, 18 (3), foy012|
|Abstract:||While 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 yeasts.|
|Rights:||Copyright © the authors,2018. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-commercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted use, distribution, and reproduction in any medium non-commercially, provided the original author and source are credited.|
|Appears in Collections:||Published Articles, Dept. of Genetics|
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