CRISPR-free, programmable RNA pseudouridylation to suppress premature termination codons

Area of Science:

• Molecular Biology
• RNA Therapeutics
• Genetic Engineering

Background:

• Nonsense mutations cause over 20% of genetic diseases by inducing premature translation termination.
• Targeting premature termination codons (PTCs) for readthrough is a potential therapeutic strategy.
• Pseudouridylation of stop codons can suppress translation termination.

Purpose of the Study:

• To develop a programmable RNA base editor, named RESTART, for reverting PTC-induced translation termination.
• To investigate the potential of RESTART as a therapeutic tool for genetic disorders.

Main Methods:

• RESTART utilizes an engineered guide snoRNA (gsnoRNA) and endogenous H/ACA box snoRNP machinery for precise pseudouridylation.
• Optimization of gsnoRNA scaffolds to enhance editing efficiency.
• Investigation of pseudouridine synthase DKC1 isoforms for improved PTC readthrough.

Main Results:

• RESTART achieved robust pseudouridylation and functional PTC readthrough in mammalian cells, including primary cells.
• A minor DKC1 isoform significantly enhanced PTC readthrough efficiency.
• Off-target pseudouridylation was limited and did not alter coding information or expression levels.

Conclusions:

• RESTART is an effective RNA base editor for precise pseudouridylation and PTC readthrough.
• The tool demonstrates potential for research and therapeutic applications in genetic diseases.
• Further development of RESTART could offer new avenues for treating nonsense mutation-related disorders.