RRC ID 49252
著者 Numamoto M, Maekawa H, Kaneko Y.
タイトル Efficient genome editing by CRISPR/Cas9 with a tRNA-sgRNA fusion in the methylotrophic yeast Ogataea polymorpha.
ジャーナル J Biosci Bioeng
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.
巻・号 124(5)
ページ 487-492
公開日 2017-11-1
DOI 10.1016/j.jbiosc.2017.06.001
PII S1389-1723(17)30214-1
PMID 28666889
MeSH CRISPR-Cas Systems / genetics* Gene Editing / methods* Genes, Fungal Genome, Fungal / genetics* Plasmids / genetics Promoter Regions, Genetic / genetics RNA, Fungal / genetics* RNA, Transfer / genetics* Saccharomycetales / genetics*
IF 2.366
引用数 17
リソース情報
酵母 BY4330