| Autor: |
Juergens H; Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629HZ Delft, The Netherlands., Varela JA; School of Microbiology/Centre for Synthetic Biology and Biotechnology/Environmental Research Institute/APC Microbiome Institute, University College Cork, Cork T12 YN60, Ireland., Gorter de Vries AR; Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629HZ Delft, The Netherlands., Perli T; Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629HZ Delft, The Netherlands., Gast VJM; Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629HZ Delft, The Netherlands., Gyurchev NY; Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629HZ Delft, The Netherlands., Rajkumar AS; School of Microbiology/Centre for Synthetic Biology and Biotechnology/Environmental Research Institute/APC Microbiome Institute, University College Cork, Cork T12 YN60, Ireland., Mans R; Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629HZ Delft, The Netherlands., Pronk JT; Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629HZ Delft, The Netherlands., Morrissey JP; School of Microbiology/Centre for Synthetic Biology and Biotechnology/Environmental Research Institute/APC Microbiome Institute, University College Cork, Cork T12 YN60, Ireland., Daran JG; Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629HZ Delft, The Netherlands. |
| Abstrakt: |
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. |
