ArticleBMC biotechnology2022
A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana.
Article in BMC biotechnology, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 23 papers.
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23 citing papers in PubMed, 44 citations in OpenAlex.
- Synthetic transcriptional repression systems in plants.Plant cell reports · 2026Review
- Causes and consequences of experimental variation in Nicotiana benthamiana transient expression.Nature communications · 2026Article
- Research progress and applications of gene activation editing technology in crops.Frontiers in plant science · 2026Review
- Synthetic promoter design in plants: integration of computational and experimental approaches.Frontiers in plant science · 2026Review
- Next-generation CRISPR systems and advanced delivery paradigms for precision crop improvement: a comparative case study in sugar beet (Frontiers in plant science · 2026Review
- Engineering Conditional Transgene Expression in Nicotiana benthamiana.Plant biotechnology journal · 2026Review
- Turbo-charging crop improvement: harnessing multiplex editing for polygenic trait engineering and beyond.The Plant journal : for cell and molecular biology · 2025Review
- Bioluminescence-Driven Optimization of Geminivirus-Based Vectors as Tools for Plant Biotechnology.ACS synthetic biology · 2025Article
- Harnessing promoter elements to enhance gene editing in plants: perspectives and advances.Plant biotechnology journal · 2025Review
- The switch-liker's guide to plant synthetic gene circuits.The Plant journal : for cell and molecular biology · 2025Review
- CuBe: a geminivirus-based copper-regulated expression system suitable for post-harvest activation.Plant biotechnology journal · 2025Article
- CRISPR activation: identifying and using novel genes for plant disease resistance breeding.Frontiers in genome editing · 2025Review
- An inducible CRISPR activation tool for accelerating plant regeneration.Plant communications · 2024Article
- Engineering Plant Cell Fates and Functions for Agriculture and Industry.ACS synthetic biology · 2024Review
- A quantitative autonomous bioluminescence reporter system with a wide dynamic range for Plant Synthetic Biology.Plant biotechnology journal · 2024Article
- Temporally gene knockout using heat shock-inducible genome-editing system in plants.The plant genome · 2023Article
- The design of synthetic gene circuits in plants: new components, old challenges.Journal of experimental botany · 2023Review
- Designing artificial synthetic promoters for accurate, smart, and versatile gene expression in plants.Plant communications · 2023Review
- Tunable control of insect pheromone biosynthesis in Nicotiana benthamiana.Plant biotechnology journal · 2023Article
- Rapid, high-titer biosynthesis of melanin using the marine bacteriumFrontiers in bioengineering and biotechnology · 2023Article
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Authors and funding
7 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundCRISPR-based programmable transcriptional activators (PTAs) are used in plants for rewiring gene networks. Better tuning of their activity in a time and dose-dependent manner should allow precise control of gene expression. Here, we report the optimization of a Copper Inducible system called CI-switch for conditional gene activation in Nicotiana benthamiana. In the presence of copper, the copper-responsive factor CUP2 undergoes a conformational change and binds a DNA motif named copper-binding site (CBS).
resultsIn this study, we tested several activation domains fused to CUP2 and found that the non-viral Gal4 domain results in strong activation of a reporter gene equipped with a minimal promoter, offering advantages over previous designs. To connect copper regulation with downstream programmable elements, several copper-dependent configurations of the strong dCasEV2.1 PTA were assayed, aiming at maximizing activation range, while minimizing undesired background expression. The best configuration involved a dual copper regulation of the two protein components of the PTA, namely dCas9:EDLL and MS2:VPR, and a constitutive RNA pol III-driven expression of the third component, a guide RNA with anchoring sites for the MS2 RNA-binding domain. With these optimizations, the CI/dCasEV2.1 system resulted in copper-dependent activation rates of 2,600-fold and 245-fold for the endogenous N. benthamiana DFR and PAL2 genes, respectively, with negligible expression in the absence of the trigger.
conclusionsThe tight regulation of copper over CI/dCasEV2.1 makes this system ideal for the conditional production of plant-derived metabolites and recombinant proteins in the field.
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