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Updated: Jun 3, 2025
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A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization
Published on: September 2, 2021
4.1K
CRISPR/Cas13X-assisted programmable and multiplexed translation regulation for controlled biosynthesis.
Xianhao Xu1,2, Xueqin Lv1,2, Yanfeng Liu1,2
1Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, No. 1800, Lihu Avenue, Binhu District, Wuxi 214122, China.
Nucleic Acids Research
|January 8, 2025
Summary
Researchers developed novel CRISPR-based tools for translational gene regulation in Bacillus subtilis, enhancing microbial cell factory efficiency. These systems offer precise control over gene expression at the translation level, improving production of valuable compounds.
Area of Science:
- Synthetic Biology
- Molecular Biology
- Microbial Biotechnology
Background:
- Gene regulation is crucial for optimizing microbial cell factories, but current tools primarily focus on transcriptional control.
- Translational regulation offers faster dynamic responses but lacks programmable, efficient, and multiplexed tools.
- Developing novel gene regulation strategies is key to advancing synthetic biology and metabolic engineering.
Purpose of the Study:
- To develop novel CRISPR-based systems for programmable gene regulation at the translational level in Bacillus subtilis.
- To engineer CRISPR interference (CRISRi) and CRISPR activation (CRISRa) tools utilizing the hfCas13X platform.
- To enhance the efficiency and applicability of these translational regulatory systems for metabolic engineering.
Main Methods:
- Construction of a CRISPR interference (CRISRi) system using catalytically deactivated hfCas13X (dhfCas13X).
- Design of unique mRNA-crRNA pairs to create degradation-inhibited CRISPRa (DiCRISPRa) and translation-started CRISPRa (TsCRISPRa) systems.
- Fusion of dhfCas13X with the RNA-binding chaperone BHfq to enhance system efficiency.
Main Results:
- Successfully developed CRISPRi and CRISPRa systems for translational gene regulation in Bacillus subtilis.
- Demonstrated significant improvement in DiCRISPRa and TsCRISPRa activation efficiency (43.2-fold) when fused with BHfq.
- Optimized metabolic networks for riboflavin and 2'-fucosyllactose production, achieving 3-fold and 1.2-fold titer increases, respectively.
Conclusions:
- The developed CRISPRa and CRISPRi systems provide novel tools for precise translational gene regulation.
- These systems offer new strategies for constructing advanced CRISPRa systems and optimizing microbial cell factories.
- The findings advance the field of gene regulation and its application in biotechnology.
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