State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China.
College of Life and Environmental Science, Shanghai Normal University, Shanghai 200234, China.
Biotechnol J. 2018 Sep;13(9):e1800121. doi: 10.1002/biot.201800121. Epub 2018 Jul 4.
Streptomycetes are Gram-positive bacteria with the capacity to produce copious bioactive secondary metabolites, which are the main source of medically and industrially relevant drugs. However, genetic manipulation of Streptomyces strains is much more difficult than other model microorganisms like Escherichia coli and Saccharomyces cerevisiae. Recently, CRISPR/Cas9 or dCas9-mediated genetic manipulation tools have been developed and facilitated Streptomyces genome editing. However, till now, CRISPR/dCas9-based interference system (CRISPRi) is only designed to repress single gene expression. Herein, the authors developed a novel CRISPRi system for multiplex gene repression in the model strain Streptomyces coelicolor. In this system, the integrative plasmid pSET152 is used as the backbone for the expression of the dCas9/sgRNA complex and both dCas9 and sgRNAs are designed to be under the control of constitutive promoters. Using the integrative CRISPRi system, the authors achieved efficient repression of multiple genes simultaneously; the mRNA levels of four targets are reduced to 2-32% of the control. Furthermore, it is successfully employed for functional gene screening, and an orphan response regulator (RR) (encoded by SCO2013) containing an RNA-binding ANTAR domain is identified being involved in bacterial growth. Collectively, this integrative CRISPRi system is very effective for multiplex gene repression in S. coelicolor, which could be extended to other Streptomyces strains for functional gene screening as well as for metabolic engineering.
链霉菌是革兰氏阳性细菌，具有产生丰富生物活性次级代谢产物的能力，这些代谢产物是具有医学和工业相关性的药物的主要来源。然而，与大肠杆菌和酿酒酵母等其他模式微生物相比，链霉菌菌株的遗传操作要困难得多。最近，CRISPR/Cas9 或 dCas9 介导的遗传操作工具已经被开发出来，并促进了链霉菌基因组编辑。然而，到目前为止，基于 CRISPR/dCas9 的干扰系统（CRISPRi）仅被设计用于抑制单个基因的表达。在此，作者开发了一种新型的 CRISPRi 系统，用于模型菌株变铅青链霉菌中的多重基因抑制。在该系统中，整合质粒 pSET152 被用作表达 dCas9/sgRNA 复合物的骨架，并且 dCas9 和 sgRNA 都被设计为受组成型启动子的控制。使用整合的 CRISPRi 系统，作者同时实现了多个基因的高效抑制；四个靶标基因的 mRNA 水平降低到对照的 2-32%。此外，该系统还成功地用于功能基因筛选，鉴定出一个含有 RNA 结合 ANTAR 结构域的孤儿响应调节子（RR）（由 SCO2013 编码）参与细菌生长。总的来说，该整合的 CRISPRi 系统在变铅青链霉菌中非常有效地进行多重基因抑制，可扩展到其他链霉菌菌株进行功能基因筛选以及代谢工程。
