Improved RNA base editing with guide RNAs mimicking highly edited endogenous ADAR substrates

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• Adenosine deaminase acting on RNA (ADAR)-mediated RNA base editing provides a reversible alternative to permanent genome editing for therapeutic applications.
• Current guide RNA (gRNA) designs, while effective, do not fully leverage the optimal substrate requirements for ADAR enzymes.
• Perfectly matched double-stranded RNA (dsRNA) is not the most efficient substrate for ADAR-mediated editing.

Purpose of the Study:

• To introduce MIRROR (mimicking inverted repeats to recruit ADARs using engineered oligoribonucleotides), a novel strategy for rational guide RNA design.
• To enhance ADAR recruitment and improve the efficiency of RNA base editing by mimicking natural, highly edited RNA structures.
• To demonstrate the applicability and superiority of the MIRROR approach in various RNA editing contexts.

Main Methods:

• Designed engineered oligoribonucleotides incorporating structural motifs from highly edited inverted Alu repeats found in human tissues.
• Tested the MIRROR approach using both chemically synthesized short gRNAs and biologically generated long gRNAs.
• Evaluated editing efficiency in multiple human cell types and primary hepatocytes from an alpha-1 antitrypsin deficiency mouse model.

Main Results:

• The MIRROR approach significantly enhances ADAR recruitment and RNA editing efficiency, achieving up to a 5.7-fold increase.
• Demonstrated the effectiveness of MIRROR with both chemically modified short gRNAs and biologically produced long gRNAs.
• Showcased improved in vitro and in vivo RNA editing capabilities in relevant cellular and animal models.

Conclusions:

• MIRROR represents a significant advancement in programmable RNA editing, offering improved efficiency and rational design principles.
• The strategy of mimicking natural, highly edited RNA substrates provides a powerful tool for optimizing ADAR-mediated RNA base editing.
• This work lays the foundation for more effective in vitro and in vivo RNA editing applications.