A method called Cas-3P allowing for immediate, multiplexed and sequential genome engineering was developed using one plasmid expressing Cas9 and three marked plasmid backbones (P1, P2 and P3) for guide RNA (gRNA) expression. The three marked gRNA plasmid backbones were recurred in a P1-P2-P3 order for sequential gene targeting, without construction of any additional plasmid and elimination of gRNA plasmid by induction in each round. The efficiency of direct gRNA plasmid curing mediated by Cas-3P was more than 40% in sequential gene targeting. Besides, Cas-3P allowed single-, double- and triple-loci gene targeting with an efficiency of 75%, 36.8% and 8.2% within 3-4 days, respectively. Through three sequential rounds of gene targeting within 10 days, S. cerevisiae was optimized for the production of patchoulol by replacing one promoter, overexpressing three genes and disrupting four genes. The work is important for practical application in the cell factory engineering of S. cerevisiae.

中文翻译：

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通过CRISPR / Cas9在酿酒酵母中进行即时，多重和顺序的基因组工程设计。

开发了一种称为Cas-3P的方法，该方法允许使用一种表达Cas9的质粒和三个用于指导RNA（gRNA）表达的标记的质粒主链（P1，P2和P3）进行即时，多重和顺序的基因组工程设计。以P1-P2-P3顺序重复三个标记的gRNA质粒骨架，以进行连续基因靶向，而无需构建任何其他质粒，也无需在每一轮中通过诱导消除gRNA质粒。在顺序基因靶向中，Cas-3P介导的直接gRNA质粒固化的效率超过40％。此外，Cas-3P可以在3-4天内分别对单，双和三位基因进行基因定位，效率分别为75％，36.8％和8.2％。通过在10天内连续进行三轮基因靶向，酿酒酵母可以通过替换一个启动子来优化广patch香酚的生产，过表达三个基因并破坏四个基因。这项工作对于酿酒酵母细胞工厂工程的实际应用很重要。