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

Hannes Juergens, Javier A Varela, Arthur R Gorter de Vries, Thomas Perli, Veronica J M Gast, Nikola Y Gyurchev, Arun S Rajkumar, Robert Mans, Jack T Pronk, John P Morrissey, Jean-Marc G Daran

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 a 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) was 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 a O. 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.