Genome editing in Kluyveromyces and Ogataea yeasts using a broad-host-range Cas9?gRNA co-expression plasmid.pdf (4.32 MB)
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Genome editing in Kluyveromyces and Ogataea yeasts using a broad-host-range Cas9/gRNA co-expression plasmid.

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journal contribution
posted on 26.09.2018, 13:38 by 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-Mark 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 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.

History

Citation

FEMS Yeast Res, 2018, 18 (3), foy012

Author affiliation

/Organisation/COLLEGE OF LIFE SCIENCES/Biological Sciences/Genetics and Genome Biology

Version

VoR (Version of Record)

Published in

FEMS Yeast Res

Publisher

Oxford University Press (OUP) for Federation of European Microbiological Societies

issn

1567-1356

eissn

1567-1364

Acceptance date

08/02/2018

Copyright date

2018

Available date

26/09/2018

Publisher version

https://academic.oup.com/femsyr/article/18/3/foy012/4847887

Language

en

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