TY - JOUR
T1 - Efficient genome editing by CRISPR/Cas9 with a tRNA-sgRNA fusion in the methylotrophic yeast Ogataea polymorpha
AU - Numamoto, Minori
AU - Maekawa, Hiromi
AU - Kaneko, Yoshinobu
N1 - Funding Information:
This work was financially supported by the Endowed Chair Funding (2011) of the Institute for Fermentation, Osaka (IFO), Japan. The authors thank the National BioResource Project Japan (YGRC/NBRP) for yeast strains and Addgene for primary CRISPR/Cas9 plasmids of S. cerevisiae .
Funding Information:
This work was financially supported by the Endowed Chair Funding (2011) of the Institute for Fermentation, Osaka (IFO), Japan. The authors thank the National BioResource Project Japan (YGRC/NBRP) for yeast strains and Addgene for primary CRISPR/Cas9 plasmids of S. cerevisiae.
Publisher Copyright:
© 2017 The Society for Biotechnology, Japan
PY - 2017/11
Y1 - 2017/11
N2 - 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 tRNACUG 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.
AB - 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 tRNACUG 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.
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U2 - 10.1016/j.jbiosc.2017.06.001
DO - 10.1016/j.jbiosc.2017.06.001
M3 - Article
C2 - 28666889
AN - SCOPUS:85021728831
SN - 1389-1723
VL - 124
SP - 487
EP - 492
JO - Journal of Bioscience and Bioengineering
JF - Journal of Bioscience and Bioengineering
IS - 5
ER -