Efficient genome editing by CRISPR/Cas9 with a tRNA-sgRNA fusion in the methylotrophic yeast Ogataea polymorpha

The methylotrophic yeast Ogataea polymorpha (syn. Hansenula polymorpha) is an attractive industrial non-conventional yeast showing high thermo-tolerance (up to 50°C) and xylose assimilation. However, genetic manipulation of O. polymorpha is often laborious and time-consuming because it has lower homologous recombination efficiency relative to Saccharomyces cerevisiae. To overcome this disadvantage, we applied the CRISPR/Cas9 system as a powerful genome editing tool in O. polymorpha. In this system, both single guide RNA (sgRNA) and endonuclease Cas9 were expressed by a single autonomously-replicable plasmid and the sgRNA portion could be easily changed by using PCR and In-Fusion cloning techniques. Because the mutation efficiency of the CRISPR/Cas9 system was relatively low when the sgRNA was expressed under the control of the OpSNR6 promoter, the tRNA^CUG gene was used for sgRNA expression. The editing efficiency of this system ranged from 17% to 71% of transformants in several target genes tested (ADE12, PHO1, PHO11, and PHO84). These findings indicate that genetic manipulation of O. polymorpha will be more convenient and accelerated by using this CRISPR/Cas9 system.