Ogataea parapolymorpha CBS11895
Ogataea parapolymorpha
CRISPR/Cas9 has enabled genome editing in a wide range of microorganisms. However, these tools are species-specific and not directly transferrable between organisms. The increasing use of non-conventional yeasts in industry has created a need for widely applicable editing tools. We present a new genome-editing tool based on the expression of ribozyme-flanked gRNAs. The gRNA and cas9 were expressed from a plasmid utilizing constitutive promoters and a pangenomic replication origin. The system was tested to knockout ADE2 in several Kluyveromyces and Ogataea (Hansenula) yeasts. In Kluyveromyces species, near-perfect targeting and homologous repair with a 960-bp DNA fragment was observed in at least 23% of the transformants. In Ogataea species no mutants were obtained directly after transformation, but prolonged liquid incubation resulted in targeting efficiencies ranging from 9 to 63%, allowing gene disruption by non-homologous end joining. The system was applied to construct an O. parapolymorpha ku80 knockout which allowed targeted deletion of the OpADE2 locus after liquid incubation at efficiencies below 1%. While prevalence of non-homologous end joining over homologous recombination prevented HR-mediatede genome editing in Ogataea, the system enabled targeted editing in Kluyveromyces and might prove useful to explore Cas9-mediated genome editing in other Saccharomycotina yeasts.