An ADAR2-mimic base editor for efficient C-to-U RNA editing in vivo.

Area of Science:

• Molecular Biology
• Genetic Engineering
• Biotechnology

Background:

• RNA base editing offers reversible and tunable genetic modification.
• Cytosine-to-uridine editors face challenges with DNA mutagenicity and immunogenicity.
• ADAR2-mimic base editor for C-to-U RNA editing (AMBER) was developed to overcome these limitations.

Purpose of the Study:

• To develop a novel RNA base editor for efficient and safe C-to-U RNA editing.
• To evaluate the performance and safety of AMBER in cellular and in vivo models.
• To assess AMBER's potential for therapeutic applications in genetic diseases.

Main Methods:

• Engineered ADAR2 into a cytosine deaminase using 17 mutant substitutions (RESCUE-S derived).
• Tested AMBER's editing efficiency and specificity in various cell lines and in C57BL/6 mice.
• Utilized RNA sequencing to analyze off-target effects and transcriptome-wide editing.

Main Results:

• AMBER demonstrated robust C-to-U editing with efficiencies from 8% to 38% in cell lines.
• Successfully corrected pathogenic mutations, including a phenylketonuria mouse model (19.7% efficiency).
• Achieved ~21% on-target efficiency in vivo in mice, with sustained editing for up to 1 week and minimal off-target effects.

Conclusions:

• AMBER is a potent RNA base editor for C-to-U conversion, overcoming limitations of previous editors.
• AMBER shows significant potential as a therapeutic tool for correcting T-to-C mutations in genetic disorders.
• The study highlights AMBER's safety and efficacy for in vivo applications in treating genetic diseases.