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Updated: Jun 23, 2025
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Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins
Published on: October 18, 2022
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Tunable translation-level CRISPR interference by dCas13 and engineered gRNA in bacteria.
Giho Kim1, Ho Joon Kim1, Keonwoo Kim1
1School of Chemical and Biological Engineering, Seoul National University, Seoul, Republic of Korea.
Nature Communications
|June 22, 2024
Summary
We developed a tunable translation-level CRISPR interference (Tl-CRISPRi) system for precise gene regulation. This RNA-targeting system enhances microbial cell factory applications, significantly boosting 3-hydroxypropionic acid production.
Area of Science:
- Molecular Biology
- Synthetic Biology
- Metabolic Engineering
Background:
- CRISPR-dCas13 systems offer translation-level gene modulation but lack precise control.
- Detailed characterization is needed to optimize RNA-guided RNA-binding protein applications.
Purpose of the Study:
- To develop a synthetic tunable translation-level CRISPR interference (Tl-CRISPRi) system.
- To enable precise and predictable down-regulation of mRNA translation.
- To optimize gene regulation in microbial systems.
Main Methods:
- Engineered guide RNAs for Tl-CRISPRi system optimization.
- Comparative analysis with transcription-level CRISPRi (Tx-CRISPRi) in polycistronic operons.
- Guide RNA handle structure engineering for tunable repression in E. coli and V. natriegens.
Main Results:
- Optimized Tl-CRISPRi for specific and multiplexed gene repression at the translation level.
- Demonstrated Tl-CRISPRi superiority over Tx-CRISPRi for polycistronic operon regulation.
- Achieved a 14.2-fold increase in 3-hydroxypropionic acid production by redirecting metabolic flux.
Conclusions:
- The tunable Tl-CRISPRi system provides precise control over mRNA translation.
- This RNA-targeting system is valuable for metabolic flux optimization in microbial cell factories.
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