CRISPRi-based circuits to control gene expression in plants

Area of Science:

• Synthetic Biology
• Plant Biotechnology
• Molecular Biology

Background:

• Synthetic gene circuit construction in plants is hindered by a scarcity of adaptable and independent components.
• Existing methods lack the orthogonality and modularity required for complex genetic programming.

Purpose of the Study:

• To establish a CRISPR interference (CRISPRi)-based, reversible gene circuit platform in plants.
• To engineer a toolkit of repressible promoters and logic gates for sophisticated gene expression control.

Main Methods:

• Implementation of a CRISPRi system with engineered repressible promoters in Arabidopsis thaliana protoplasts.
• Development of NOT and NOR logic gates and optimization of single-guide RNA expression.
• Testing of CRISPRi gate functionality in stably transformed Arabidopsis and cross-species protoplasts (Physcomitrium patens, Triticum aestivum, Brassica napus).

Main Results:

• Successful construction and demonstration of NOR gates in Arabidopsis protoplasts and whole plants, showcasing programmability and reversibility.
• Validation of CRISPRi logic gate activity across multiple plant species.
• Creation of OR, NIMPLY, and AND logic functions by integrating multiple NOR gates, confirming system modularity.

Conclusions:

• The developed CRISPRi platform provides orthogonal, compact, reversible, programmable, and modular synthetic gene circuits for plants.
• This system enables sophisticated spatiotemporal control of gene expression, advancing plant synthetic biology.