A robust dual gene ON-OFF toggle directed by two independent promoter-degron pairs

Area of Science:

• Molecular Biology
• Gene Regulation
• Cell Biology

Background:

• Understanding gene function is crucial, often requiring precise control over gene expression.
• CRISPR-mediated knockout combined with rescue constructs is used for loss-of-function studies of essential genes.
• Simultaneously activating a second gene is needed to interrogate pathway functions.

Purpose of the Study:

• To develop a novel system for independently controlling gene switching (ON/OFF) in mammalian cell lines.
• To enable simultaneous toggling between two constructs with comparable kinetics and tightness.
• To facilitate functional studies of essential genes and pathways.

Main Methods:

• Developed a gene-OFF switch using TRE transcriptional control and auxin-induced degron-mediated proteolysis.
• Engineered an independent gene-ON switch utilizing a modified ecdysone promoter and a destabilization domain degron.
• Created a two-gene switch platform for tightly regulated gene activation and inactivation.

Main Results:

• Successfully created independently controlled gene-ON and gene-OFF switches.
• Achieved comparable kinetics and tightness in toggling between constructs.
• Demonstrated acute and tuneable gene activation and inactivation within a cell cycle.

Conclusions:

• The developed two-gene switch platform allows for efficient generation of knockout cell lines.
• This system provides tight regulation and rapid switching of gene activity.
• Enables advanced functional studies of gene interactions and pathways.