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

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Juergens, Hannes
Varela, Javier A.
Gorter de Vries, Arthur R.
Perli, Thomas
Gast, Veronica J. M.
Gyurchev, Nikola Y.
Rajkumar, Arun S.
Mans, Robert
Pronk, Jack T.
Morrissey, John P.
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Oxford University Press
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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
Crispr/cas9 , Ribozymes , Kluyveromyces lactis , Kluyveromyces marxianus , Ogataea polymorpha , Hansenula polymorpha
Juergens, 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/foy012