Tunable translation-level CRISPR interference by dCas13 and engineered gRNA in bacteria

Giho Kim, Ho Joon Kim, Keonwoo Kim, Hyeon Jin Kim, Jina Yang and Sang Woo Seo ()
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Giho Kim: Seoul National University
Ho Joon Kim: Seoul National University
Keonwoo Kim: Seoul National University
Hyeon Jin Kim: Seoul National University
Jina Yang: Jeju National University
Sang Woo Seo: Seoul National University

Nature Communications, 2024, vol. 15, issue 1, 1-13

Abstract: 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.

Date: 2024
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DOI: 10.1038/s41467-024-49642-x

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