Overcoming Leak Sensitivity in CRISPRi Circuits Using Antisense RNA Sequestration and Regulatory Feedback

Area of Science:

• Synthetic biology
• Molecular biology
• Genetic engineering

Background:

• CRISPR interference (CRISPRi) utilizes catalytically dead CRISPR nuclease (dCas) for programmable gene regulation.
• Existing dCas-based genetic circuits face limitations from leaky repression and shared dCas protein pools, impacting performance.

Purpose of the Study:

• To develop a mechanism to suppress unwanted CRISPRi repression and enhance logical gene circuit function.
• To improve the performance of dCas-based genetic circuits, particularly during stationary expression and in layered circuit designs.

Main Methods:

• Utilized antisense RNAs (asRNAs) to sequester guide RNA (gRNA) transcripts.
• Implemented CRISPRi feedback for self-regulation of asRNA production.
• Analyzed circuit induction at the single-cell level using microfluidic channels.

Main Results:

• Demonstrated a mechanism that effectively suppresses unwanted CRISPRi repression.
• Showed significant improvement in logical gene circuit function in Escherichia coli.
• Restored logical performance in layered circuits, such as double inverters, using dual CRISPRi/asRNA inverters.
• Provided single-cell level insights into antisense RNA sequestration and regulatory feedback dynamics.

Conclusions:

• The developed dual CRISPRi/asRNA system enhances the reliability and performance of dCas-based genetic circuits.
• This approach overcomes key limitations of CRISPRi, enabling more complex and robust synthetic biology applications.
• The study highlights the potential for improved CRISPRi inverters in advanced genetic circuitry without compromising dCas programmability.