Optimizing a CRISPR-Cas13d Gene Circuit for Tunable Target RNA Downregulation with Minimal Collateral RNA Cutting

Area of Science:

• Molecular Biology
• Biotechnology
• Gene Editing

Background:

• RNA-guided RNA cutting systems, such as Cas13d, offer alternatives to RNA interference for transcriptome engineering.
• "Collateral damage" from off-target RNA cutting has limited the enthusiasm for these systems.
• The dependence of RNA reduction and collateral activity on Cas13d and guide RNA abundance was previously unclear.

Purpose of the Study:

• To investigate how Cas13d and guide RNA levels influence both specific (on-target) and nonspecific RNA reduction.
• To develop novel gene circuits for precise control over RNA-guided RNA cutting systems.
• To minimize collateral activity while maximizing on-target RNA reduction for biotechnological and therapeutic applications.

Main Methods:

• Utilized precise expression-tuning gene circuits to modulate Cas13d and guide RNA levels.
• Developed new Multi-Level Optimized Negative-Autoregulated Cas13d and crRNA Hybrid (MONARCH) gene circuits.
• Assessed RNA reduction and collateral activity in human kidney cells and green monkey cells.

Main Results:

• Both on-target and nonspecific RNA reduction were found to be dependent on Cas13d and guide RNA abundance.
• Nonspecific RNA cutting (trans cleavage) may contribute to on-target RNA reduction.
• MONARCH gene circuits demonstrated a high dynamic range with low basal on-target RNA reduction and minimized collateral activity.

Conclusions:

• Precise control over Cas13d and guide RNA levels is crucial for optimizing RNA-guided RNA cutting systems.
• MONARCH gene circuits offer a solution to mitigate collateral activity and improve the applicability of these systems.
• These advancements position RNA-guided RNA cutting as a powerful, programmable tool for transcriptome engineering in biotechnology and medicine.