Molecular Cell. 2023 Jan 5
Jinghui Song, Liting Dong, Hanxiao Sun, Nan Luo, Qiang Huang, Kai Li, Xiaowen Shen, Zhe Jiang, Zhicong Lv, Luxin Peng, Meifang Zhang, Kun Wang, Ke Liu, Jiaxu Hong, Chengqi Yi
Products used in the paper Details Operation
AAV vector packaging: AAV6.2 (EEF2-U1570); AAV6.2 (EEF2-U2881); AAV6.2 (PPIB-U272); AAV6.2 (CFTR-W1282X); AAVDJ (mIdua-W392X) RESTARTv2 expressing AAVs were packaged in PackGene Company. Request Quote

Research Field: RNA editing

AAV Serotype: AAV6.2; AAVDJ

Animal or cell line strain: HDF and HBE cells were seeded at density of 3 x 10^5 in T75 flask, B-Lymphocytes were seeded at density of 2 x 10^5/ml, and MEFs were seeded into 100 mm culture dishes at a density of 1.5 x 10^5 cells. 24 hours after seeding, cells were transduced by AAV6.2 (EEF2- U1570/EEF2- 2881/PPIB-U272/CFTR-W1282X) at multiplicities of infection 1 x 10^6 (for HDF), 1 x 10^5 (for HBE), and 1 x 10^5 (for B-Lymphocytes). MEFs were transduced by AAVDJ (Idua-W392X) at multiplicities of infection 5 x 10^6. Cell medium were changed 24 hours after transduction. 7 days after transduction, cells were collected.


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.

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