Inducing circular RNA formation using the CRISPR endoribonuclease Csy4

Area of Science:

• Molecular Biology
• Synthetic Biology
• RNA Biology

Background:

• Circular RNAs (circRNAs) are stable, covalently closed RNA molecules with largely unknown functions.
• circRNAs hold potential for engineered systems and technological applications.
• Regulating circRNA formation is crucial for understanding their roles.

Purpose of the Study:

• To develop a novel switch for inducing back-splicing of engineered circRNAs.
• To utilize the CRISPR endoribonuclease Csy4 as an activator for circRNA circularization.
• To establish a platform for regulating splicing and studying circRNA properties.

Main Methods:

• Engineered a circRNA switch responsive to CRISPR Csy4.
• Coexpressed Csy4 with the circRNA switch to activate circularization.
• Leveraged Csy4's endoribonuclease activity to remove competing splice sites.
• Stabilized the 5' cleavage product for subsequent back-splicing.

Main Results:

• Demonstrated successful induction of back-splicing using the Csy4-activated circRNA switch.
• Showcased the stabilization of the 5' cleavage product by Csy4.
• Achieved translation of a reporter protein from the engineered circRNA via an IRES.
• Established a straightforward approach for regulating circRNA splicing.

Conclusions:

• The Csy4-activated circRNA switch provides a robust method for controlled circRNA formation.
• This platform offers potential applications in synthetic biology and fundamental circRNA research.
• The system enables studying circRNA properties and engineering novel RNA-based systems.