CRISPR-Cas Expands Dynamic Range of Gene Expression From T7RNAP Promoters
| Autor: | Sean R. McCutcheon, Daniel D. Lewis, Kwan Lun Chiu, Cheemeng Tan |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
030106 microbiology Gene Expression Computational biology Applied Microbiology and Biotechnology law.invention Viral Proteins 03 medical and health sciences Synthetic biology law Gene expression medicine Protein biosynthesis CRISPR T7 RNA polymerase Promoter Regions Genetic Gene Editing Genetics biology Promoter DNA-Directed RNA Polymerases General Medicine biology.organism_classification 030104 developmental biology Recombinant DNA Molecular Medicine Synthetic Biology CRISPR-Cas Systems Bacteria medicine.drug |
| Zdroj: | Biotechnology Journal. 13:1700167 |
| ISSN: | 1860-6768 |
| DOI: | 10.1002/biot.201700167 |
| Popis: | Reducing leaky gene expression is critical for improving protein yield of recombinant bacteria and stability of engineered cellular circuits in synthetic biology. Leaky gene expression occurs when a genetic promoter is not fully repressed, leading to unintended protein synthesis in the absence of stimuli. Existing work have devised specific molecular strategies for reducing leaky gene expression of each promoter. In contrast, we describe a repurposed, modular CRISPRi system that attenuates leaky gene expression using a series of single-guide RNAs targeting the PT7/LacO1 promoter. Furthermore, we demonstrate the efficacy of CRISPRi to significantly increase the dynamic range of T7 RNA Polymerase (T7RNAP) promoters. In addition, we demonstrate that the CRISPRi system can be applied to enhance growth of bacteria that suffer from leaky expression of a toxic protein. Our work establishes a new application of CRISPRi in genomic engineering to improve the control of recombinant gene expression. The approach is potentially generalizable to other gene expression system by changing the single-guide RNAs. |
| Databáze: | OpenAIRE |
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