CRISPR-assisted multi-dimensional regulation for fine-tuning gene expression in Bacillus subtilis
CRISPR-assisted multi-dimensional regulation for fine-tuning gene expression in Bacillus subtilis
复制标题
CRISPR辅助多维调控枯草芽孢杆菌基因表达的微调
DOI：
10.1093/nar/gkz072
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发表时间：
2019-04-23
影响因子：
14.9
通讯作者：
Zhang, Guimin
中科院分区：
文献类型：
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作者：
Lu, Zhenghui;Yang, Shihui;Zhang, Guimin
摘要基因表达的微调对于蛋白质的表达和通路的构建是至关重要的，但由于在多个水平上以上下文相关的方式进行基因分级调控，它仍然面临着巨大的挑战。在本研究中，我们定义了dCas9-α/ω系统中CRISPRA和CRISPRI的最佳靶向窗口，并证明了该系统可以作为单一的主调控因子，通过设计位置特异的gRNA来同时激活和抑制不同基因的表达。基于ω的启动子改组(OAPS)策略进一步扩大了dCas9-DCAS9的应用范围，该策略可以产生高比例的功能启动子突变，有利于在转化效率较低的微生物中构建有效的启动子文库。结合OAPs和dCas9-ω，系统地研究了启动子转录、分子伴侣辅助的蛋白质折叠和蛋白酶介导的降解对枯草芽孢杆菌淀粉酶BLA表达的影响，使BLA的产量提高了260倍。OAPs策略和dCas9-ω在本研究中用于BLA生产的成功表明，它可以作为一个强大的工具包来多方向和多维度地调控细菌中多基因的表达。
Abstract Fine-tuning of gene expression is crucial for protein expression and pathway construction, but it still faces formidable challenges due to the hierarchical gene regulation at multiple levels in a context-dependent manner. In this study, we defined the optimal targeting windows for CRISPRa and CRISPRi of the dCas9-α/ω system, and demonstrated that this system could act as a single master regulator to simultaneously activate and repress the expression of different genes by designing position-specific gRNAs. The application scope of dCas9-ω was further expanded by a newly developed CRISPR-assisted Oligonucleotide Annealing based Promoter Shuffling (OAPS) strategy, which could generate a high proportion of functional promoter mutants and facilitate the construction of effective promoter libraries in microorganisms with low transformation efficiency. Combing OAPS and dCas9-ω, the influences of promoter-based transcription, molecular chaperone-assisted protein folding and protease-mediated degradation on the expression of amylase BLA in Bacillus subtilis were systematically evaluated, and a 260-fold enhancement of BLA production was obtained. The success of the OAPS strategy and dCas9-ω for BLA production in this study thus demonstrated that it could serve as a powerful tool kit to regulate the expression of multiple genes multi-directionally and multi-dimensionally in bacteria.