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CRISPRi-based circuits to control gene expression in plants

Nature Biotechnology (2024)Cite this article

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Abstract

The construction of synthetic gene circuits in plants has been limited by a lack of orthogonal and modular parts. Here, we implement a CRISPR (clustered regularly interspaced short palindromic repeats) interference (CRISPRi)-based reversible gene circuit platform in plants. We create a toolkit of engineered repressible promoters of different strengths and construct NOT and NOR gates in Arabidopsis thaliana protoplasts. We determine the optimal processing system to express single guide RNAs from RNA Pol II promoters to introduce NOR gate programmability for interfacing with host regulatory sequences. The performance of a NOR gate in stably transformed Arabidopsis plants demonstrates the system’s programmability and reversibility in a complex multicellular organism. Furthermore, cross-species activity of CRISPRi-based logic gates is shown in Physcomitrium patens, Triticum aestivum and Brassica napus protoplasts. Layering multiple NOR gates together creates OR, NIMPLY and AND logic functions, highlighting the modularity of our system. Our CRISPRi circuits are orthogonal, compact, reversible, programmable and modular and provide a platform for sophisticated spatiotemporal control of gene expression in plants.

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Fig. 1: Designing and testing integrators for a plant CRISPRi–dCas9-based NOR gate.
Fig. 2: Synthetic promoter evaluation, NOR gate construction and sgRNA processing.
Fig. 3: dCas9–repressor fusions in plant gene circuits.
Fig. 4: In vivo circuit performance in transgenic Arabidopsis plants and protoplasts of different species.
Fig. 5: Programmable multilayered circuits.
Fig. 6: Effect of input sgRNA promoter strength on single-layered and two-layered circuit performance.

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Data availability

All the data analyzed and used for generating figures in this study are provided in Supplementary Tables 5 and 8. The plasmids generated in this study will be made available through Addgene. DNA sequences are available in a Zenodo repository79 (https://doi.org/10.5281/zenodo.10528436).

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Acknowledgements

The plasmid sequencing data were generated on instrumentation supported by the Australian Cancer Research Foundation Center for Advanced Cancer Genomics and Genomics WA. Seeds for the LhGR-N line were kindly provided by C. Helliwell. This work was supported by the following grants to R.L.: Australian Research Council (ARC) Center of Excellence (CoE) in plant energy biology (CE140100008), ARC CoE in plants for space (CE230100015), ARC DP210103954, NHMRC Investigator Grant GNT1178460, Silvia and Charles Viertel Senior Medical Research Fellowship and Howard Hughes Medical Institute International Research Scholarship. M.A.K. was supported by an International Postgraduate Research Scholarship. T.S. was supported by the Hackett Postgraduate Research Scholarship. B.K. was supported by the CSIRO Synthetic Biology Future Science Platform. D.S. was supported by an ARC Discovery Early Career Researcher Award (DE150100460).

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Authors and Affiliations

  1. Australian Research Council Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Perth, Western Australia, Australia

    Muhammad Adil Khan, Gabrielle Herring, Jia Yuan Zhu, Marina Oliva, Elliott Fourie, Benjamin Johnston, Zhining Zhang, Jarred Potter, Luke Pineda, Christian Pflueger, James P. B. Lloyd, David Secco, Ian Small, Brendan N. Kidd & Ryan Lister

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

    Muhammad Adil Khan, Gabrielle Herring, Jia Yuan Zhu, Marina Oliva, Elliott Fourie, James P. B. Lloyd, Ian Small & Ryan Lister

  3. Harry Perkins Institute of Medical Research, The University of Western Australia, Perth, Western Australia, Australia

    Jahnvi Pflueger, Tessa Swain, Christian Pflueger & Ryan Lister

  4. CSIRO Synthetic Biology Future Science Platform, Brisbane, Queensland, Australia

    Brendan N. Kidd

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Contributions

M.A.K., B.N.K. and R.L. conceptualized the study, designed the experiments and wrote the manuscript. M.A.K., with B.N.K., G.H., J.Y.Z., M.O., E.F., B.J., Z.Z., J.P., L.P. and J.P.B.L., conducted the experiments. M.A.K. designed the constructs and developed the 96-well protoplast transfection protocol. J.P., T.S. and C.P. conducted the plasmid sequencing. J.P.B.L, D.S. and I.S. provided assistance with the initial designs of constructs and designing experiments. All authors approved of and contributed to the final version of the manuscript.

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Khan, M.A., Herring, G., Zhu, J.Y. et al. CRISPRi-based circuits to control gene expression in plants. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02236-w

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