PDF(Pubmed)
Abstract：
A large number of recombinant plasmids for the yeast Saccharomyces cerevisiae have been constructed and accumulated over the past four decades. It is desirable to apply the recombinant plasmid resources to Saccharomyces sensu stricto species group, which contains an increasing number of natural isolate and industrial strains. The application to the group encounters a difficulty. Natural isolates and industrial strains are exclusively prototrophic and polyploid, whereas direct application of most conventional plasmid resources imposes a prerequisite in host yeast strains of an auxotrophic mutation (i.e., leu2) that is rescued by a selection gene (e.g., LEU2) on the recombinant plasmids. To solve the difficulty, we aimed to generate leu2 mutants from yeast strains belonging to the yeast Saccharomyces sensu stricto species group by DNA editing. First, we modified an all-in-one type CRISPR-Cas9 plasmid pML104 by adding an antibiotic-resistance gene and designing guide sequences to target the LEU2 gene and to enable wide application in this yeast group. Then, the resulting CRISPR-Cas9 plasmids were exploited to seven strains belonging to five species of the group, including natural isolate, industrial, and allopolyploid strains. Colonies having the designed mutations in the gene appeared successfully by introducing the plasmids and assisting oligonucleotides to the strains. Most of the plasmids and resultant leu2- mutants produced in this study will be deposited in several repository organizations. KEY POINTS: • All-in-one type CRISPR-Cas9 plasmids targeting LEU2 gene were designed for broad application to Saccharomyces sensu stricto group species strains • Application of the plasmids generated leu2 mutants from strains including natural isolates, industrial, and allopolyploid strains • The easy conversion to leu2 mutants permits free access to recombinant plasmids having a LEU2 gene.
摘要：
在过去的四十年中，已经构建并积累了大量用于酿酒酵母的重组质粒。希望将重组质粒资源应用于严格的酵母物种组，其中包含越来越多的天然分离株和工业菌株。对组的应用遇到困难。天然分离株和工业菌株完全是原养型和多倍体，而直接应用大多数常规质粒资源在宿主酵母菌株中施加了营养缺陷型突变的先决条件(即，leu2)由选择基因(例如，LEU2)在重组质粒上。为了解决困难，我们旨在通过DNA编辑从属于严格的酵母酵母物种组的酵母菌株中生成leu2突变体。首先,我们通过添加抗生素抗性基因并设计针对LEU2基因的指导序列,修饰了多合一型CRISPR-Cas9质粒pML104,并使其能够在该酵母组中广泛应用.然后，所得的CRISPR-Cas9质粒被利用到属于该组的五个物种的七个菌株中，包括天然分离物,工业,和异源多倍体菌株。在基因中具有设计突变的集落通过将质粒和辅助寡核苷酸引入菌株而成功地出现。本研究中产生的大多数质粒和所得的leu2-突变体将存放在几个存储库组织中。关键词：•靶向LEU2基因的一体化CRISPR-Cas9质粒被设计用于广泛地应用于严格意义的酵母群物种菌株•质粒的应用从包括天然分离物的菌株中产生了leu2突变体,工业,和异源多倍体菌株•容易转化为leu2突变体允许自由获得具有LEU2基因的重组质粒。
