Numamoto Minori, Maekawa Hiromi, Kaneko Yoshinobu
Yeast Genetic Resources Laboratory, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Yeast Genetic Resources Laboratory, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
J Biosci Bioeng. 2017 Nov;124(5):487-492. doi: 10.1016/j.jbiosc.2017.06.001. Epub 2017 Jun 27.
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 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.
甲基营养型酵母多形奥塔酵母（同义词：多形汉逊酵母）是一种具有吸引力的工业非常规酵母，表现出较高的耐热性（高达50°C）和木糖同化能力。然而，多形奥塔酵母的基因操作通常费力且耗时，因为相对于酿酒酵母，它的同源重组效率较低。为了克服这一缺点，我们将CRISPR/Cas9系统作为一种强大的基因组编辑工具应用于多形奥塔酵母。在该系统中，单导向RNA（sgRNA）和核酸内切酶Cas9均由单个自主复制质粒表达，并且sgRNA部分可通过使用PCR和In-Fusion克隆技术轻松改变。由于当sgRNA在OpSNR6启动子控制下表达时，CRISPR/Cas9系统的突变效率相对较低，因此使用tRNA基因进行sgRNA表达。在测试的几个靶基因（ADE12、PHO1、PHO11和PHO84）中，该系统的编辑效率在转化体的17%至71%之间。这些发现表明，使用这种CRISPR/Cas9系统将使多形奥塔酵母的基因操作更加便捷和快速。
