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

Nonsense mutations, accounting for >20% of disease-associated mutations, lead to premature translation termination. Replacing uridine with pseudouridine in stop codons suppresses translation termination, which could be harnessed to mediate readthrough of premature termination codons (PTCs). Here, we present RESTART, a programmable RNA base editor, to revert PTC-induced translation termination in mammalian cells. RESTART utilizes an engineered guide snoRNA (gsnoRNA) and the endogenous H/ACA box snoRNP machinery to achieve precise pseudouridylation. We also identified and optimized gsnoRNA scaffolds to increase the editing efficiency. Unexpectedly, we found that a minor isoform of pseudouridine synthase DKC1, lacking a C-terminal nuclear localization signal, greatly improved the PTC-readthrough efficiency. Although RESTART induced restricted off-target pseudouridylation, they did not change the coding information nor the expression level of off-targets. Finally, RESTART enables robust pseudouridylation in primary cells and achieves functional PTC readthrough in disease-relevant contexts. Collectively, RESTART is a promising RNA-editing tool for research and therapeutics. [Display omitted] •RESTART relies on guide snoRNA to revert PTC-induced translation termination•The minor DKC1-isoform3 greatly increases the RNA-editing efficiency of RESTART•RESTART enables efficient pseudouridylation in cell lines and primary cells•RESTART mediates functional PTC readthrough in disease-relevant contexts Song et al. developed a programmable RNA base editor, termed RESTART, relying on guide snoRNA to mediate site-specific pseudouridylation in mammalian cells. RESTART was applied to nonsense mutations in multiple disease contexts to achieve functional PTC readthrough.