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

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

Jürgens, H. (TU Delft BT/Industriele Microbiologie)
Varela, Javier A. (University College Cork)
Gorter de Vries, A.R. (TU Delft BT/Industriele Microbiologie)
Perli, T. (TU Delft BT/Industriele Microbiologie)
Gast, V.J.M. (Student TU Delft)
Gyurchev, N.Y. (Student TU Delft)
Rajkumar, Arun S. (University College Cork)
Mans, R. (TU Delft BT/Industriele Microbiologie)
Pronk, J.T. (TU Delft BT/Biotechnologie)
Morrissey, John P. (University College Cork)
Daran, J.G. (TU Delft BT/Industriele Microbiologie)

BT/Biotechnologie

2018

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
Hansenula polymorpha
Kluyveromyces lactis
Kluyveromyces marxianus
Ogataea polymorpha
Ribozymes

http://resolver.tudelft.nl/uuid:8acf4570-d02b-4ee4-997f-ac709ed6daa3

https://doi.org/10.1093/femsyr/foy012

1567-1356

FEMS Yeast Research, 18 (3)

Institutional Repository

journal article

© 2018 H. Jürgens, Javier A. Varela, A.R. Gorter de Vries, T. Perli, V.J.M. Gast, N.Y. Gyurchev, Arun S. Rajkumar, R. Mans, J.T. Pronk, John P. Morrissey, J.G. Daran