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Updated: Jun 4, 2026
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Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins
Published on: October 18, 2022
Programmable, multiplexed and orthogonal gene control in bacteria with attenuated Cas13d systems
Shengkun Tong1, Yuxi Qin1, Yaqian Sun1
1State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
Nature Biotechnology
|June 2, 2026
Summary
Engineered Cas13d variants offer tunable RNA knockdown in bacteria, reducing cytotoxicity. This versatile toolkit enables precise gene regulation for synthetic biology and biotechnology applications.
Area of Science:
- Microbial synthetic biology
- RNA-based biotechnology
- CRISPR gene editing
Background:
- Cas13-based RNA effectors offer dynamic gene regulation but face challenges with cytotoxicity and collateral cleavage.
- Existing systems require optimization for safe and effective use in bacterial applications.
Purpose of the Study:
- To engineer attenuated Cas13d variants with reduced toxicity and tunable RNase activity.
- To develop a versatile RNA-regulatory system for programmable gene control in bacteria.
- To demonstrate the application of this system for optimizing metabolic pathways.
Main Methods:
- Rational protein engineering involving targeted truncation of flexible Cas13d regions.
- Introduction of proximal mismatches in CRISPR RNA spacers for functional switching.
- Multiplexed gene regulation within polycistronic mRNAs and synthetic circuits.
- Application in optimizing lycopene biosynthesis in Escherichia coli.
Main Results:
- Generated attenuated Cas13d variants with significantly reduced cytotoxicity (2.2-fold higher growth).
- Achieved tunable transcript knockdown and functional switching between translation inhibition and mRNA degradation.
- Demonstrated programmable, orthogonal, and multiplexed gene regulation.
- Successfully optimized lycopene biosynthesis, improving yields through pathway rewiring.
Conclusions:
- Engineered Cas13d variants provide a safer and more versatile platform for bacterial RNA regulation.
- The developed system enables precise control over gene expression in complex synthetic circuits.
- This RNA-regulatory toolkit advances microbial synthetic biology and RNA-based biotechnology.
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