Targeted RNA base editing for therapeutic: mechanisms and advances

Fig. 1. Overview of RNA base editors and elements of RNA base editing systems. A. A-to-I, C-to-U, A-to-m6A/m6A-to-A, and U-to-Ψ base editors are gradually developed recent years, but there exist other unexploited but potential base conversions for RNA base editing. B. A general RNA base editor mainly contains targeting system (e.g., Cas systems), effector protein (e.g., ADAR, exogenous or endogenous). Besides, delivery system is also vital for the medical application of RNA editors.

Fig. 2. Targeting systems of RNA base editors: (1) RNP based targeting systems. Cas targeting system is the most used targeting system, SNAP-tag is a chemical tag for tying protein and RNA together. (2) Protein/RNA-alone targeting systems. PUF targeting system is a protein-alone targeting system, while (H/ACA box) snoRNA targeting system is RNA-alone.

Fig. 3. Base conversion, modification patterns and effector proteins of developed RNA base editors. A. A-to-I (read as G). B. C-to-U. C. U-to-Ψ. D. A-to-m6A/m6A-to-A.

Fig. 4. Exogenous ADAR for A-to-I RNA base editing. A. Diagram of SNAP-ADAR and improved SNAP-ADAR. A SNAP-tag was fused to ADAD DD, and it reacted with BG-modified gRNA, to form gRNA–deaminase conjugates. B. Diagram of Cas-ADAR. A dCas protein was fused to ADAR DD to direct the editing of targeted cytosine.

Fig. 5. Endogenous ADAR for A-to-I RNA base editing. A. Diagram of RESTORE. Chemical modified gRNA with ADAR recruiting domain. B. Diagram of LEAPER. Engineered long linear RNA-targeting domain only. C. Diagram of LEAPER 2.0. Engineered circular RNA-targeting domain with covalently closed loop. D. Diagram of CLUSTER. Add a cluster of single-stranded recruitment sequences on R/G motif gRNA.

Fig. 6. C-to-U editors for RNA base editing. A. Diagram of RESCUE. An evolved ADAR DD tethered to a dCas13b. B. Diagram of CURE. An APOBEC3A protein tethered to a dCasRx. C. Diagram of CU-REWIRE. An evolved ADAR DD tethered to a 10-repeats PUF-domain.

Fig. 7. A-to-m6A/m6A-to-A base editors and U-to-Ψ editors. A. Diagram of A-to-m6A/m6A-to-A base editors. A m6A demethylase or adenosine methylase tethered to a dCas protein. B. Diagram of TRADES. A dCas13b tethered to SunTag system, and 10 copies of GCN4 peptide enable recruited the scFv-fusion m6A demethylase. C. Diagram of enhanced or reversible m6A editing by red/far-red light induction. Red light triggered ΔphyA-dCas13 recruitment of FHY1-fused METTL3 and A-to-m6A editing, Far-red light induced Bphp1-FTO recruitment via PspR2-MCP and m6A-to-A editing. D. Diagram of RESTART and Targeted pseudouridylation. They used engineered or over-expressed H/ACA box snoRNA for the site-specific pseudouridylation on PTCs to suppress the nonsense mutations.