Precise in vivo RNA base editing with a wobble-enhanced circular CLUSTER guide RNA.

Area of Science:

• Molecular Biology
• Genetic Engineering
• RNA Therapeutics

Background:

• Adenosine deaminase acting on RNA (ADAR) enables adenosine-to-inosine (A-to-I) RNA base editing for genetic manipulation.
• Off-target bystander editing remains a significant challenge, limiting the precision and safety of current RNA base-editing systems.

Purpose of the Study:

• To develop a strategy to mitigate bystander off-target editing in A-to-I RNA base editing.
• To enhance the precision and efficiency of RNA base editing for potential therapeutic applications.

Main Methods:

• Investigated the role of G•U wobble base pairs in 5'-UAN triplets, common sites for bystander editing.
• Applied G•U wobble base pairing strategy in conjunction with the CLUSTER approach (circularized format).
• Utilized virus-mediated delivery of guide RNA for in vivo studies in a murine model.

Main Results:

• G•U wobble base pairs effectively suppress off-target editing at 5'-UAN sites while maintaining high on-target efficiency.
• Combined wobble base pairing and circularized CLUSTER approach achieved up to 87% editing efficiency for a Mecp2 mutation in cell culture.
• In vivo delivery in a murine Rett syndrome model restored MeCP2 protein function with up to 19% editing yield and controlled bystander effects.

Conclusions:

• G•U wobble base pairing is a universally applicable strategy to enhance the precision of A-to-I RNA base editing.
• This method complements existing strategies and significantly improves the safety profile of RNA base editing.
• Demonstrated the potential of this enhanced RNA editing system for treating genetic disorders like Rett syndrome.