CRISPRi-based Circuits to Control Gene Expression in Plants

Cell Biology Bioengineering Biochemistry Botany Life Sciences
Gene Editing CRISPRi Synthetic Biology Logic Gates Plant Biotechnology Gene Expression

Plant Gene Expression Control Circuit Based on CRISPRi

Academic Background

In the field of plant biotechnology, traditional gene manipulation methods focus on producing the desired phenotypes and cellular activities through continuous transgene expression. However, strong continuous promoters may lead to gene silencing, metabolic burdens, or other adverse effects on yield, preventing the full realization of the potential benefits of transgenes. Through synthetic biology methods, constructing synthetic gene circuits is expected to address these challenges. Synthetic gene circuits enhance spatiotemporal control of gene expression by integrating multiple input signals to control gene output.

Authors and Source

This study comes from multiple researchers at the School of Molecular Sciences, University of Western Australia, including Muhammad Adil Khan, Gabrielle Herring, Jia Yuan Zhu, and others. It was published in the journal Nature Biotechnology, with the submission date of July 29, 2022, the acceptance date of April 10, 2024, and for the publication date and online updates, please refer to the article.

Research Process

  1. The research team first selected Arabidopsis thaliana’s Translationally Controlled Tumor Protein (TCTP) as the initial target to develop a reversible gene circuit platform based on CRISPR interference (CRISPRi).
  2. By constructing engineered promoters of different strengths, they created NOT and NOR logic gates and determined the optimal handling system for expressing single guide RNAs (sgRNAs) from RNA Pol II promoters, achieving NOR gate programming and the interface with biological host regulatory sequences.
  3. They demonstrated the programmability and reversibility of the system by showing the performance of NOR gates in stably transformed Arabidopsis plants and established the activity of cross-species logic gates in various Arabidopsis protoplasts.
  4. By layering multiple NOR gates, they created OR, NIMPLY, and AND logic functions, demonstrating the system’s modularity.

Main Results

a) The study shows that programmable and reversible synthetic gene circuits can be constructed in plant cells using CRISPRi. b) The developed synthetic promoters can be effectively used to control gene expression and build logic gates. c) It was demonstrated that CRISPRi-based logic gates are active in multiple plant species, including Arabidopsis thaliana, the moss Physcomitrium patens, wheat (Triticum aestivum), and rapeseed (Brassica napus). d) By connecting multiple NOR gates, they constructed complex logic functions such as OR, NIMPLY, and AND.

Conclusion and Implications

a) Scientific Value: This provides a new platform for plant synthetic biology to achieve complex programmable control of gene expression, aiding in the programming of plant responses to dynamic internal and external environmental stimuli. b) Application Value: This technology is expected to improve plants’ ability to cope with environmental stress, increase yields, and provide new tools for future crop improvement.

Research Highlights

1) The study introduces the functionality of CRISPRi logic gates and their applicability in various plant species, expanding the application field of synthetic biology in plant genetic engineering. 2) Research Methods and Workflow: Developed a high-throughput protoplast transfection testing platform, increasing testing throughput and reducing the interference of transfection rate variability on the analysis of logic gate functions. 3) The provided toolkit (including engineered promoters and synthetic gene circuits) is beneficial for researchers to develop more advanced spatiotemporal control gene expression strategies.