CRISPR-free Programmable RNA pseudouridylation to suppress premature termination codons

We present RESTART, a programmable approach employing engineered artificial guide snoRNA (gsnoRNA) to achieve precise and efficient site-directed pseudouridylation in mammalian cells without changing the coding information. We identified and optimized gsnoRNA scaffolds, which can mediate PTC-readthrough by recruiting endogenous DKC1 (pseudouridine synthase). We also found combining exogenous non-canonical DKC1-isoform3 with gsnoRNA can further improve the efficiency of PTC-readthrough. We applied such strategies to correct disease-relevant nonsense mutations, while maintaining the global ? abundance in cellular RNA and gene expression. Collectively, RESTART would become a powerful tool for therapeutics and basic research. Overall design: Examination of HEK293T cells co-transfected of 2 µg ALDOB-W148X (or SMN1-W190X) and 2 µg gACA19 / gCtrl / empty vector (Vec) together with or without 500 ng DKC1-isoform3 (iso3) constructs. Detection of pseudouridine modification by CeU-seq and targeted deep amplicon sequencing