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- Title
Tunable translation-level CRISPR interference by dCas13 and engineered gRNA in bacteria.
- Authors
Kim, Giho; Kim, Ho Joon; Kim, Keonwoo; Kim, Hyeon Jin; Yang, Jina; Seo, Sang Woo
- Abstract
Although CRISPR-dCas13, the RNA-guided RNA-binding protein, was recently exploited as a translation-level gene expression modulator, it has still been difficult to precisely control the level due to the lack of detailed characterization. Here, we develop a synthetic tunable translation-level CRISPR interference (Tl-CRISPRi) system based on the engineered guide RNAs that enable precise and predictable down-regulation of mRNA translation. First, we optimize the Tl-CRISPRi system for specific and multiplexed repression of genes at the translation level. We also show that the Tl-CRISPRi system is more suitable for independently regulating each gene in a polycistronic operon than the transcription-level CRISPRi (Tx-CRISPRi) system. We further engineer the handle structure of guide RNA for tunable and predictable repression of various genes in Escherichia coli and Vibrio natriegens. This tunable Tl-CRISPRi system is applied to increase the production of 3-hydroxypropionic acid (3-HP) by 14.2-fold via redirecting the metabolic flux, indicating the usefulness of this system for the flux optimization in the microbial cell factories based on the RNA-targeting machinery. It's difficult to precisely modulate translation-level gene expression using CRISPR-dCas13 due to the lack of detailed characterization of the system. Here, the authors fill the knowledge gap and develop a synthetic tunable Tl-CRISPRi system to enable precise and predictable control of mRNA translation.
- Subjects
CRISPRS; GENE expression; RNA-binding proteins; GENETIC translation; OPERONS; MICROBIAL cells
- Publication
Nature Communications, 2024, Vol 15, Issue 1, p1
- ISSN
2041-1723
- Publication type
Article
- DOI
10.1038/s41467-024-49642-x