Journal
NUCLEIC ACIDS RESEARCH
Volume 47, Issue 7, Pages -Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkz072
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Funding
- National Natural Science Foundation of China [31670069]
- Technical Innovation Special Fund of Hubei Province [2017ACA171]
- 2016 Wuhan Yellow Crane Talents (Science) Program
- Distinguished Young Scholars of Hubei Province [2018CFA042]
- State Key Laboratory of Biocatalysis and Enzyme Engineering
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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-/ 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- 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-, 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- 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.
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