State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, Hubei, China 430062.
Nucleic Acids Res. 2019 Apr 23;47(7):e40. doi: 10.1093/nar/gkz072.
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.
基因表达的精细调控对于蛋白质表达和途径构建至关重要，但由于在上下文相关的情况下，多层次的基因调控具有层次性，因此仍然面临着巨大的挑战。在本研究中，我们定义了 CRISPRa 和 CRISPRi 的 dCas9-α/ω 系统的最佳靶向窗口，并通过设计位置特异性 gRNA 证明该系统可以作为单个主调节剂同时激活和抑制不同基因的表达。通过新开发的基于 CRISPR 的寡核苷酸退火启动子改组（OAPS）策略，进一步扩展了 dCas9-ω 的应用范围，该策略可以生成高比例的功能性启动子突变体，并促进在转化效率低的微生物中构建有效的启动子文库。通过将 OAPS 和 dCas9-ω 相结合，系统评估了基于启动子的转录、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中淀粉酶 BLA 表达的影响，获得了 260 倍的 BLA 产量提高。本研究中 OAPS 策略和 dCas9-ω 在 BLA 生产中的成功，证明了它们可以作为一种强大的工具包，在细菌中多向、多维地调控多个基因的表达。
