A nuclease-dead Cas9-derived tool represses target gene expression

Area of Science:

• Plant molecular biology
• Gene regulation and epigenetics
• Biotechnology and synthetic biology

Background:

• Precise control of gene expression is crucial for understanding gene function and crop improvement.
• Nuclease-dead Cas9 (dCas9) systems offer targeted genome manipulation without DNA cleavage.
• An effective and universal dCas9-based transcriptional repression system for plants is currently lacking.

Purpose of the Study:

• To engineer and validate a novel dCas9-based transcriptional repression system for plants.
• To identify the key functional domain of TENDRIL-LESS (TEN) for gene repression.
• To enhance the genetic regulation toolkit for precision breeding in crops.

Main Methods:

• Fusion of dCas9 with full-length and truncated TEN proteins from cucumber (Cucumis sativus).
• Validation of gene repression efficacy and specificity in cucumber and Arabidopsis thaliana via transient infection and genetic transformation.
• Electrophoretic mobility shift assay (EMSA) to investigate DNA-binding properties of the TEN N-terminal domain.

Main Results:

• The dCas9-TEN fusion protein effectively repressed target gene expression in plants.
• The N-terminal domain of TEN was identified as the core repression domain.
• EMSA confirmed the TEN N-terminal domain's ability to bind chromatin, potentially enhancing dCas9 targeting and repression.

Conclusions:

• A novel and effective dCas9-TEN transcriptional repression system has been developed for plants.
• This tool provides enhanced precision for gene regulation in plant biotechnology.
• The findings contribute to the development of advanced genetic tools for crop precision breeding.