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Designing and testing CRISPRi-based synthetic gene circuits in plants

Nature Protocols (2026)Cite this article

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Abstract

Synthetic gene circuits are powerful tools for precisely programming gene expression and introducing novel cellular functions. However, their development and application in plants has lagged behind other systems, due mainly to the limited availability of modular genetic parts. We recently developed a CRISPR interference (CRISPRi)-based synthetic gene circuit system for programming gene expression in plants. Using a robust and high-throughput protoplast-based dual luciferase assay, we demonstrated the development, testing and functionality of these circuits in various plant species. Here we detail the key design principles and considerations for building and testing programmable and reversible CRISPRi-based gene circuits in plants. We also provide detailed procedures for isolating protoplasts from multiple plant species, including Arabidopsis thaliana, Brassica napus, Triticum aestivum and Physcomitrium patens. Furthermore, we provide step-by-step instructions for the 96-well plate-based protoplast transfection assay for testing genetic parts and synthetic circuits, using a dual luciferase assay. The detailed descriptions of these developed systems will enhance the efficiency and reproducibility of the construction, testing, and implementation of synthetic gene circuits in a variety of plant species. This protocol enables the design and testing of CRISPRi-based gene circuits in plants within ~4 weeks.

Key points

  • The protocol describes design principles for CRISPRi-based genetic circuits in plants, and outlines procedures for isolating protoplasts from different plant species and tissues, and for testing genetic parts and gene circuits using a plate-based, high-throughput, dual luciferase protoplast transfection assay.

  • CRISPRi-based circuits can integrate different signals to achieve programmable gene expression in plants, while the high-throughput protoplast-based assay permits rapid and scalable testing of circuit components across different species.

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Fig. 1: Workflow for testing synthetic gene circuits in plants.
Fig. 2: NOR logic and components of a CRISPRi-based gene circuit system in plants.
Fig. 3: Design of the sensor and integrator modules.
Fig. 4: Anticipated results of plant optimal growth stages and isolated protoplasts.
Fig. 5: Anticipated results of CRISPRi-based gene circuits in various plant species.

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Data availability

All data presented in this protocol is available in the primary supporting paper4, except for Fig. 5d, which is provided as Source data. The DNA sequences for the no-input and input sgRNA-A constructs used to generate Fig. 5d are the exact same constructs used in Fig. 3h of the primary supporting paper. All plasmid DNA sequences used in this protocol are available in the Zenodo repository36, and have also been deposited to Addgene for public access under plasmid IDs 239869-239946. Source data are provided with this paper.

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Acknowledgements

This work was supported by the following grants to R.L.: Australian Research Council (ARC) Center of Excellence in Plants for Space (CE230100015), ARC Discovery Project grants (DP210103954 and DP240103385), and National Health and Medical Research Council Investigator Grants (GNT1178460 and GNT2035042).

Author information

Authors and Affiliations

  1. Australian Research Council Centre of Excellence in Plants for Space, School of Molecular Sciences, The University of Western Australia, Perth, Western Australia, Australia

    Adil Khan, Gabrielle Herring, Jia Yuan Zhu, Milly Petterson & Ryan Lister

  2. Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Perth, Western Australia, Australia

    Ryan Lister

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Contributions

A.K. and R.L. designed the study, conceived and developed the CRISPRi-based gene circuits system and wrote the manuscript. A.K. optimized the procedure for isolating protoplasts from A. thaliana leaves, and established the 96-well protoplast transfection protocol. A.K. and G.H. optimized protoplast isolation from T. aestivum and B. napus leaves, while G.H. and M.P. refined the protocol for isolating protoplasts from A. thaliana roots. J.Y.Z. optimized protoplast isolation from P. patens. All authors reviewed, contributed to and approved the final manuscript.

Corresponding author

Correspondence to Ryan Lister.

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Nature Protocols thanks Alexander Leydon and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Key references

Khan, M. A., Herring, G., Zhu, J. Y. et al. Nat. Biotechnol. 43, 416–430 (2025): https://doi.org/10.1038/s41587-024-02236-w

Lloyd, J. P. B., Ly, F., Gong, P. et al. Nat. Biotechnol. 40, 1862–1872 (2022): https://doi.org/10.1038/s41587-022-01383-2

Supplementary information

Supplementary Information (download PDF )

Supplementary Figs. 1–3, Video 1 and Description of stock solutions.

Reporting Summary (download PDF )

Supplementary Video 1 (download MOV )

Leaf peeling procedure for protoplast isolation.

Source data

Source Data Fig. 5 (download XLSX )

Statistical source data for Fig. 5d.

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Khan, A., Herring, G., Zhu, J.Y. et al. Designing and testing CRISPRi-based synthetic gene circuits in plants. Nat Protoc (2026). https://doi.org/10.1038/s41596-025-01312-y

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