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Efficient genome editing by CRISPR/Cas9 with a tRNA-sgRNA fusion in the methylotrophic yeast Ogataea polymorpha
- Source :
- Journal of Bioscience and Bioengineering. 124:487-492
- Publication Year :
- 2017
- Publisher :
- Elsevier BV, 2017.
-
Abstract
- 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.
- Subjects :
- 0106 biological sciences
0301 basic medicine
Genes, Fungal
Saccharomyces cerevisiae
Bioengineering
01 natural sciences
Applied Microbiology and Biotechnology
03 medical and health sciences
Plasmid
RNA, Transfer
Genome editing
010608 biotechnology
CRISPR
Guide RNA
Promoter Regions, Genetic
Gene Editing
Genetics
biology
Cas9
RNA, Fungal
biology.organism_classification
030104 developmental biology
Saccharomycetales
CRISPR-Cas Systems
Genome, Fungal
Ogataea polymorpha
Homologous recombination
Plasmids
Biotechnology
Subjects
Details
- ISSN :
- 13891723
- Volume :
- 124
- Database :
- OpenAIRE
- Journal :
- Journal of Bioscience and Bioengineering
- Accession number :
- edsair.doi.dedup.....22364d20b42c434d888c4b9e2e80dc74