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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).

References

  1. Thompson, A. J. et al. Ectopic expression of a tomato 9-cis-epoxycarotenoid dioxygenase gene causes over-production of abscisic acid. Plant J. 23, 363–374 (2000).

    Article  CAS  PubMed  Google Scholar 

  2. Iuchi, S. et al. Regulation of drought tolerance by gene manipulation of 9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis. Plant J. 27, 325–333 (2001).

    Article  CAS  PubMed  Google Scholar 

  3. Feeney, M., Frigerio, L., Cui, Y. & Menassa, R. Following vegetative to embryonic cellular changes in leaves of Arabidopsis overexpressing LEAFY COTYLEDON2. Plant Physiol. 162, 1881–1896 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Vanhercke, T. et al. Step changes in leaf oil accumulation via iterative metabolic engineering. Metab. Eng. 39, 237–246 (2017).

    Article  CAS  PubMed  Google Scholar 

  5. He, R. et al. Overexpression of 9-cis-epoxycarotenoid dioxygenase cisgene in grapevine increases drought tolerance and results in pleiotropic effects. Front. Plant Sci. 9, 970 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Hancock, J. F. A framework for assessing the risk of transgenic crops. Bioscience 53, 512–519 (2003).

    Article  Google Scholar 

  7. Jaglo, K. R. et al. Components of the Arabidopsis C-repeat/dehydration-responsive element binding factor cold-response pathway are conserved in Brassica napus and other plant species. Plant Physiol. 127, 910–917 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Brophy, J. A. N. & Voigt, C. A. Principles of genetic circuit design. Nat. Methods 11, 508–520 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kassaw, T. K., Donayre-Torres, A. J., Antunes, M. S., Morey, K. J. & Medford, J. I. Engineering synthetic regulatory circuits in plants. Plant Sci. 273, 13–22 (2018).

    Article  CAS  PubMed  Google Scholar 

  10. Andres, J., Blomeier, T. & Zurbriggen, M. D. Synthetic switches and regulatory circuits in plants. Plant Physiol. 179, 862–884 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. de Lange, O., Klavins, E. & Nemhauser, J. Synthetic genetic circuits in crop plants. Curr. Opin. Biotechnol. 49, 16–22 (2018).

    Article  PubMed  Google Scholar 

  12. Xia, P.-F., Ling, H., Foo, J. L. & Chang, M. W. Synthetic genetic circuits for programmable biological functionalities. Biotechnol. Adv. 37, 107393 (2019).

    Article  PubMed  Google Scholar 

  13. Verbič, A., Praznik, A. & Jerala, R. A guide to the design of synthetic gene networks in mammalian cells. FEBS J. 288, 5265–5288 (2021).

    Article  PubMed  Google Scholar 

  14. Chen, Y. et al. Genetic circuit design automation for yeast. Nat. Microbiol. 5, 1349–1360 (2020).

    Article  CAS  PubMed  Google Scholar 

  15. Weber, W. & Fussenegger, M. Engineering of synthetic mammalian gene networks. Chem. Biol. 16, 287–297 (2009).

    Article  CAS  PubMed  Google Scholar 

  16. Lienert, F., Lohmueller, J. J., Garg, A. & Silver, P. A. Synthetic biology in mammalian cells: next generation research tools and therapeutics. Nat. Rev. Mol. Cell Biol. 15, 95–107 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Mahfouz, M. M. et al. Targeted transcriptional repression using a chimeric TALE–SRDX repressor protein. Plant Mol. Biol. 78, 311–321 (2012).

    Article  CAS  PubMed  Google Scholar 

  18. Lowder, L. G., Paul, J. W. 3rd & Qi, Y. Multiplexed transcriptional activation or repression in plants using CRISPR–dCas9-based systems. Methods Mol. Biol. 1629, 167–184 (2017).

    Article  CAS  PubMed  Google Scholar 

  19. Hiratsu, K., Matsui, K., Koyama, T. & Ohme-Takagi, M. Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis. Plant J. 34, 733–739 (2003).

    Article  CAS  PubMed  Google Scholar 

  20. Leydon, A. R. et al. Repression by the Arabidopsis TOPLESS corepressor requires association with the core mediator complex. eLife 10, e66739 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Leydon, A. R., Ramos Báez, R. & Nemhauser, J. L. A single helix repression domain is functional across diverse eukaryotes. Proc. Natl Acad. Sci. USA 119, e2206986119 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Vazquez-Vilar, M. et al. GB3.0: a platform for plant bio-design that connects functional DNA elements with associated biological data. Nucleic Acids Res. 45, 2196–2209 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Schaumberg, K. A. et al. Quantitative characterization of genetic parts and circuits for plant synthetic biology. Nat. Methods 13, 94 (2015).

    Article  PubMed  Google Scholar 

  24. Brophy, J. A. N. et al. Synthetic genetic circuits as a means of reprogramming plant roots. Science 377, 747–751 (2022).

    Article  CAS  PubMed  Google Scholar 

  25. Belcher, M. S. et al. Design of orthogonal regulatory systems for modulating gene expression in plants. Nat. Chem. Biol. 16, 857–865 (2020).

    Article  CAS  PubMed  Google Scholar 

  26. Bernabé-Orts, J. M. et al. A memory switch for plant synthetic biology based on the phage ϕC31 integration system. Nucleic Acids Res. 48, 3379–3394 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  27. Lloyd, J. P. B. et al. Synthetic memory circuits for stable cell reprogramming in plants. Nat. Biotechnol. 40, 1862–1872 (2022).

    Article  CAS  PubMed  Google Scholar 

  28. Guiziou, S., Maranas, C. J., Chu, J. C. & Nemhauser, J. L. An integrase toolbox to record gene-expression during plant development. Nat. Commun. 14, 1844 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Gander, M. W., Vrana, J. D., Voje, W. E., Carothers, J. M. & Klavins, E. Digital logic circuits in yeast with CRISPR–dCas9 NOR gates. Nat. Commun. 8, 15459 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Kiani, S. et al. CRISPR transcriptional repression devices and layered circuits in mammalian cells. Nat. Methods 11, 723–726 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Yeo, N. C. et al. An enhanced CRISPR repressor for targeted mammalian gene regulation. Nat. Methods 15, 611–616 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Nielsen, A. A. K. & Voigt, C. A. Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks. Mol. Syst. Biol. 10, 763 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Santos-Moreno, J., Tasiudi, E., Stelling, J. & Schaerli, Y. Multistable and dynamic CRISPRi-based synthetic circuits. Nat. Commun. 11, 2746 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Kim, H., Bojar, D. & Fussenegger, M. A CRISPR/Cas9-based central processing unit to program complex logic computation in human cells. Proc. Natl Acad. Sci. USA 116, 7214–7219 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Jones, D. L. et al. Kinetics of dCas9 target search in Escherichia coli. Science 357, 1420–1424 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Martens, K. J. A. et al. Visualisation of dCas9 target search in vivo using an open-microscopy framework. Nat. Commun. 10, 3552 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Santos-Moreno, J. & Schaerli, Y. CRISPR-based gene expression control for synthetic gene circuits. Biochem. Soc. Trans. 48, 1979–1993 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Lowder, L. G. et al. A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol. 169, 971–985 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Piatek, A. et al. RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors. Plant Biotechnol. J. 13, 578–589 (2015).

    Article  CAS  PubMed  Google Scholar 

  41. Vazquez-Vilar, M. et al. A modular toolbox for gRNA–Cas9 genome engineering in plants based on the GoldenBraid standard. Plant Methods 12, 10 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Vazquez-Vilar, M. et al. The GB4.0 platform, an all-in-one tool for CRISPR/Cas-based multiplex genome engineering in plants. Front. Plant Sci. 12, 689937 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Khakhar, A., Leydon, A. R., Lemmex, A. C., Klavins, E. & Nemhauser, J. L. Synthetic hormone-responsive transcription factors can monitor and re-program plant development. eLife 7, e34702 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Han, Y.-J., Kim, Y.-M., Hwang, O.-J. & Kim, J.-I. Characterization of a small constitutive promoter from Arabidopsis translationally controlled tumor protein (AtTCTP) gene for plant transformation. Plant Cell Rep. 34, 265–275 (2015).

    Article  CAS  PubMed  Google Scholar 

  45. Somssich, M. A short history of the CaMV 35S promoter. Preprint at PeerJ https://doi.org/10.7287/peerj.preprints.27096v3 (2019).

  46. Yilmaz, A. et al. AGRIS: the Arabidopsis Gene Regulatory Information Server, an update. Nucleic Acids Res. 39, D1118–D1122 (2011).

    Article  CAS  PubMed  Google Scholar 

  47. Davuluri, R. V. et al. AGRIS: Arabidopsis gene regulatory information server, an information resource of Arabidopsis cis-regulatory elements and transcription factors. BMC Bioinformatics 4, 25 (2003).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Palaniswamy, S. K. et al. AGRIS and AtRegNet. A platform to link cis-regulatory elements and transcription factors into regulatory networks. Plant Physiol. 140, 818–829 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Vancanneyt, G., Schmidt, R., O’Connor-Sanchez, A., Willmitzer, L. & Rocha-Sosa, M. Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol. Gen. Genet. 220, 245–250 (1990).

    Article  CAS  PubMed  Google Scholar 

  50. Gao, Y. & Zhao, Y. Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. J. Integr. Plant Biol. 56, 343–349 (2014).

    Article  CAS  PubMed  Google Scholar 

  51. Cermak, T. et al. A multi-purpose toolkit to enable advanced genome engineering in plants. Plant Cell 29, 1196–1217 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Xie, K., Minkenberg, B. & Yang, Y. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc. Natl Acad. Sci. USA 112, 3570–3575 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Haurwitz, R. E., Jinek, M., Wiedenheft, B., Zhou, K. & Doudna, J. A. Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science 329, 1355–1358 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Nissim, L., Perli, S. D., Fridkin, A., Perez-Pinera, P. & Lu, T. K. Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Mol. Cell 54, 698–710 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Lodish, H., et al. Molecular Cell Biology (W. H. Freeman, 2000).

  56. Schramm, L. & Hernandez, N. Recruitment of RNA polymerase III to its target promoters. Genes Dev. 16, 2593–2620 (2002).

    Article  CAS  PubMed  Google Scholar 

  57. Sherf, B. A., Navarro, S. L., Hannah, R. R. & Wood, K. V. Dual-luciferase TM reporter assay: an advanced co-reporter technology integrating firefly and Renilla luciferase assays. Promega Notes 57, 2–8 (1996).

    Google Scholar 

  58. McNabb, D. S., Reed, R. & Marciniak, R. A. Dual luciferase assay system for rapid assessment of gene expression in Saccharomyces cerevisiae. Eukaryot. Cell 4, 1539–1549 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Gilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Gilbert, L. A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Thakore, P. I., Black, J. B., Hilton, I. B. & Gersbach, C. A. Editing the epigenome: technologies for programmable transcription and epigenetic modulation. Nat. Methods 13, 127–137 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Craft, J. et al. New pOp/LhG4 vectors for stringent glucocorticoid-dependent transgene expression in Arabidopsis. Plant J. 41, 899–918 (2005).

    Article  CAS  PubMed  Google Scholar 

  63. Takahashi, T., Naito, S. & Komeda, Y. The Arabidopsis HSP18.2 promoter/GUS gene fusion in transgenic Arabidopsis plants: a powerful tool for the isolation of regulatory mutants of the heat‐shock response. Plant J. 2, 751–761 (1992).

    Article  CAS  Google Scholar 

  64. Horstmann, V., Huether, C. M., Jost, W., Reski, R. & Decker, E. L. Quantitative promoter analysis in Physcomitrella patens: a set of plant vectors activating gene expression within three orders of magnitude. BMC Biotechnol. 4, 13 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  65. Gaber, R. et al. Designable DNA-binding domains enable construction of logic circuits in mammalian cells. Nat. Chem. Biol. 10, 203–208 (2014).

    Article  CAS  PubMed  Google Scholar 

  66. Leben, K. et al. Binding of the transcription activator-like effector augments transcriptional regulation by another transcription factor. Nucleic Acids Res. 50, 6562–6574 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Tompa, M. et al. Assessing computational tools for the discovery of transcription factor binding sites. Nat. Biotechnol. 23, 137–144 (2005).

    Article  CAS  PubMed  Google Scholar 

  68. Jiang, C. & Pugh, B. F. Nucleosome positioning and gene regulation: advances through genomics. Nat. Rev. Genet. 10, 161–172 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Heard, D. J., Kiss, T. & Filipowicz, W. Both Arabidopsis TATA binding protein (TBP) isoforms are functionally identical in RNA polymerase II and III transcription in plant cells: evidence for gene-specific changes in DNA binding specificity of TBP. EMBO J. 12, 3519–3528 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Mukumoto, F., Hirose, S., Imaseki, H. & Yamazaki, K. DNA sequence requirement of a TATA element-binding protein from Arabidopsis for transcription in vitro. Plant Mol. Biol. 23, 995–1003 (1993).

    Article  CAS  PubMed  Google Scholar 

  71. Gorochowski, T. E. et al. Genetic circuit characterization and debugging using RNA-seq. Mol. Syst. Biol. 13, 952 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  72. Ohta, M., Matsui, K., Hiratsu, K., Shinshi, H. & Ohme-Takagi, M. Repression domains of class II ERF transcriptional repressors share an essential motif for active repression. Plant Cell 13, 1959–1968 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Kagale, S. & Rozwadowski, K. EAR motif-mediated transcriptional repression in plants: an underlying mechanism for epigenetic regulation of gene expression. Epigenetics 6, 141–146 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Yang, E. J. Y. & Nemhauser, J. L. Building a pipeline to identify and engineer constitutive and repressible promoters. Quant. Plant Biol. 4, e12 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  75. Tas, H., Grozinger, L., Stoof, R., de Lorenzo, V. & Goñi-Moreno, Á. Contextual dependencies expand the re-usability of genetic inverters. Nat. Commun. 12, 355 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    Article  CAS  PubMed  Google Scholar 

  77. Engler, C. et al. A Golden Gate modular cloning toolbox for plants. ACS Synth. Biol. 3, 839–843 (2014).

    Article  CAS  PubMed  Google Scholar 

  78. Pollak, B. et al. Universal loop assembly: open, efficient and cross-kingdom DNA fabrication. Synth. Biol. 5, ysaa001 (2020).

    Article  CAS  Google Scholar 

  79. Khan, M. A. et al. CRISPRi-based circuits to control gene expression in plants. Zenodo https://doi.org/10.5281/zenodo.11108565 (2024).

  80. Wick, R. R., Judd, L. M., Gorrie, C. L. & Holt, K. E. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput. Biol. 13, e1005595 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  81. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Wu, F.-H. et al. Tape–Arabidopsis Sandwich—a simpler Arabidopsis protoplast isolation method. Plant Methods 5, 16 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  83. Cove, D. J. et al. Isolation and regeneration of protoplasts of the moss Physcomitrella patens. Cold Spring Harb. Protoc. https://doi.org/10.1101/pdb.prot5140 (2009).

    Article  PubMed  Google Scholar 

  84. Cove, D. J. et al. Transformation of the moss Physcomitrella patens using direct DNA uptake by protoplasts. Cold Spring Harb. Protoc. https://doi.org/10.1101/pdb.prot5143 (2009).

    Article  PubMed  Google Scholar 

  85. Ganguly, D. R., Tyrrell, R. & Arndell, T. Protoplast isolation and PEG-mediated transformation. Protocols.io https://doi.org/10.17504/protocols.io.36wgqwd5gk57/v2 (2022).

  86. Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).

    Article  CAS  PubMed  Google Scholar 

  87. Logemann, E., Birkenbihl, R. P., Ülker, B. & Somssich, I. E. An improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transformation protocol. Plant Methods 2, 16 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

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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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