Expanding RNA editing toolkit using an IDR-based strategy

Area of Science:

• Molecular Biology
• Biotechnology
• Genetic Engineering

Background:

• RNA base editing offers precise genetic modification but faces challenges in efficiency and specificity for C to U changes.
• Existing editors often lack ideal characteristics such as being compact, efficient, and non-immunogenic.

Purpose of the Study:

• To develop a compact, fully human C to U RNA base editor.
• To enhance the efficiency of RNA base editors using a novel strategy.

Main Methods:

• Fusion of the human C to U editing enzyme RESCUE-S with the Cas inspired RNA targeting system (CIRTS) to create CIRTS-RESCUEv1 (V1).
• Incorporation of a histidine-rich domain (HRD) from human CYCT1, known for liquid-liquid phase separation (LLPS), into the V1 editor.
• Evaluation of editing efficiency and mechanism, including LLPS-dependent puncta formation.

Main Results:

• The initial V1 editor showed low efficiency.
• Fusion with HRD significantly enhanced on-target editing efficiency of V1.
• HRD-mediated enhancement was linked to liquid-liquid phase separation (LLPS), with other intrinsically disordered regions (IDRs) also showing potentiation.
• The HRD fusion strategy proved applicable to other C to U RNA editors.

Conclusions:

• A novel, compact, human-origin C to U RNA base editor (CIRTS-RESCUEv1) was developed.
• Incorporation of intrinsically disordered regions, leveraging liquid-liquid phase separation, is a general and effective strategy to enhance C to U RNA base editor efficiency.
• This work expands the toolkit for RNA editing and provides a method to improve editor performance.