RECODE: a programmable guide-free C-to-U RNA editing tool

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• CRISPR-based RNA cytidine deaminase tools offer programmable C-to-U editing but face limitations like low efficiency and off-target effects.
• Existing RNA editing systems require guide RNAs, limiting targetable sequences and flexibility.

Purpose of the Study:

• To develop a novel, guide-free RNA editing tool for efficient and specific C-to-U conversion.
• To engineer and optimize a tool for enhanced editing activity, reduced off-target effects, and broad applicability.

Main Methods:

• Engineered RECODE (RNA Editor for C-to-U with an Optimized DYW Enzyme) using RNA-binding pentatricopeptide repeat proteins fused to a DYW cytidine deaminase domain.
• Optimized RECODE's specificity domain for retargeting and reduced off-target edits, and enhanced its catalytic region for increased activity and translation.

Main Results:

• RECODE demonstrated efficient C-to-U editing in human cells across diverse targets, achieving over 50% efficiency for most sites.
• The tool showed high specificity, avoiding edits at adjacent cytidines, and proved functional in mice with high efficiency in skeletal muscle tissue via AAV delivery.

Conclusions:

• RECODE represents a significant advancement in RNA editing technology, offering a guide-free, efficient, and specific C-to-U conversion system.
• The demonstrated *in vivo* functionality and therapeutic potential suggest RECODE could be valuable for treating various diseases.