2. Academic Validation
  3. CRISPR-free, programmable RNA pseudouridylation to suppress premature termination codons

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

  • Mol Cell. 2022 Dec 3;S1097-2765(22)01100-5. doi: 10.1016/j.molcel.2022.11.011.
Jinghui Song 1,  Liting Dong 2,  Hanxiao Sun 1,  Nan Luo 1,  Qiang Huang 1,  Kai Li 3,  Xiaowen Shen 4,  Zhe Jiang 1,  Zhicong Lv 1,  Luxin Peng 5,  Meifang Zhang 6,  Kun Wang 1,  Ke Liu 7,  Jiaxu Hong 8,  Chengqi Yi 9
Affiliations

Affiliations

  • 1 State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, PRC.
  • 2 State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, PRC; Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, PRC.
  • 3 State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, PRC; Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, PRC; Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, PRC.
  • 4 Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, PRC.
  • 5 College of Chemistry and Molecular Engineering, Peking University, Beijing, PRC.
  • 6 Elpis Biotechnology Company Limited, Beijing, PRC.
  • 7 Department of Otolaryngology Head and Neck Surgery, Beijing Friendship Hospital, Capital Medical University, Beijing, PRC.
  • 8 Department of Ophthalmology, Eye and Ear, Nose, Throat Hospital of Fudan University, Shanghai, PRC.
  • 9 State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, PRC; Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, PRC; Department of Chemical Biology and Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, Beijing, PRC. Electronic address: [email protected].
PMID: 36521489 DOI: 10.1016/j.molcel.2022.11.011
Abstract

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.

Keywords

DKC1; PTC readthrough; RNA base editor; RNA editing; RNA modification; RNA targeting; nonsense mutation; premature termination codon; pseudourine; snoRNA.

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