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Designing and testing CRISPRi-based synthetic gene circuits in plants
Adil Khan1, Gabrielle Herring1, Jia Yuan Zhu1
1Australian Research Council Centre of Excellence in Plants for Space, School of Molecular Sciences, The University of Western Australia, Perth, Western Australia, Australia.
Nature Protocols
|February 4, 2026
Summary
We developed a new CRISPR interference (CRISPRi)-based synthetic gene circuit system for programming gene expression in plants. This protocol enhances the efficiency and reproducibility of synthetic biology applications in diverse plant species.
Area of Science:
- Synthetic biology
- Plant biotechnology
- Gene expression regulation
Background:
- Synthetic gene circuits offer precise control over gene expression and novel cellular functions.
- Development of these circuits in plants is limited by the availability of modular genetic parts.
Purpose of the Study:
- To detail the design, construction, and testing of CRISPR interference (CRISPRi)-based synthetic gene circuits for plants.
- To provide a robust protocol for evaluating genetic parts and circuits in various plant species.
Main Methods:
- Utilized a CRISPR interference (CRISPRi) system for gene expression programming.
- Employed a high-throughput, protoplast-based dual luciferase assay for testing.
- Developed detailed procedures for protoplast isolation and transfection in multiple plant species (Arabidopsis thaliana, Brassica napus, Triticum aestivum, Physcomitrium patens).
Main Results:
- Demonstrated the functionality of CRISPRi-based synthetic gene circuits in various plant species.
- Established a 96-well plate-based assay for efficient testing of genetic parts and circuits.
- The protocol enables the design and testing of plant gene circuits within approximately 4 weeks.
Conclusions:
- The developed protocol enhances the efficiency and reproducibility of synthetic gene circuit construction and testing in plants.
- This system facilitates the implementation of programmable and reversible gene expression control in diverse plant species.
- Advances plant synthetic biology by providing modular tools for precise genetic programming.
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