Blue Light-Operated CRISPR/Cas13b-Mediated mRNA Knockdown (Lockdown)

Area of Science:

• Molecular Biology
• Optogenetics
• Gene Regulation

Background:

• Optogenetics offers precise spatial, temporal, and dynamic light control over gene expression at transcriptional and protein stability levels.
• Current RNA downregulation strategies like RNA interference and CRISPR/Cas methods lack optical control advantages.
• A need exists for light-inducible RNA knockdown tools with high specificity and control.

Purpose of the Study:

• To introduce a novel optogenetic system, "Lockdown," for light-controlled RNA level regulation.
• To demonstrate the combination of "Lockdown" with other optogenetic tools for multi-level gene downregulation.
• To investigate the application of "Lockdown" in controlling cell cycle progression.

Main Methods:

• Developed "Lockdown," a system using blue light to induce CRISPR/Cas13b expression for sequence-specific mRNA knockdown.
• Combined "Lockdown" with optogenetic tools for transcriptional repression and protein destabilization.
• Applied "Lockdown" to target endogenous cyclin-dependent kinase 1 (hCdk1) mRNA in mammalian cells.

Main Results:

• "Lockdown" successfully achieved blue light-induced, sequence-specific mRNA knockdown.
• Combining "Lockdown" with other optogenetic tools resulted in efficient triple-controlled protein downregulation.
• Degradation of hCdk1 mRNA using "Lockdown" induced G2/M cell cycle arrest and inhibited cell growth.

Conclusions:

• "Lockdown" provides a powerful new optogenetic strategy for precise RNA level control.
• This system enables multi-modal light-driven gene silencing, offering enhanced research capabilities.
• "Lockdown" demonstrates potential for controlling cellular processes like cell cycle progression.