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Near-cognate tRNAs increase the efficiency and precision of pseudouridine-mediated readthrough of premature termination codons

Nature Biotechnology (2024)Cite this article

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Abstract

Programmable RNA pseudouridylation has emerged as a new type of RNA base editor to suppress premature termination codons (PTCs) that can lead to truncated and nonfunctional proteins. However, current methods to correct disease-associated PTCs suffer from low efficiency and limited precision. Here we develop RESTART v3, which uses near-cognate tRNAs to improve the readthrough efficiency of pseudouridine-modified PTCs. We show an average of ~5-fold (range: 2.1- to 9.5-fold) higher editing efficiency than RESTART v2 in cultured cells and achieve functional PTC readthrough in disease cell models of cystic fibrosis and Hurler syndrome. Furthermore, RESTART v3 enables accurate incorporation of the original amino acid for nearly half of the PTC sites, considering the naturally occurring frequencies of sense-to-nonsense codons, without affecting normal termination codons. Although off-target sites were detected, we did not observe changes to the coding information or the expression level of transcripts, and the overall natural tRNA abundance remained constant.

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Fig. 1: Near-cognate tRNAs improve the readthrough efficiency of RESTART.
Fig. 2: Screening of near-cognate tRNAs to improve readthrough efficiency of all three PTCs.
Fig. 3: RESTART v3 can achieve accurate correction of PTC sites.
Fig. 4: Characterizing the specificity of RESTART v3.
Fig. 5: Functional PTC readthrough in CFTR and IDUA disease cell models.

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Data availability

The sequence data generated in this study have been deposited in the NCBI Gene Expression Omnibus (GEO), under accession code GSE237633 (ref. 57). Source data are provided with this paper.

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Acknowledgements

The authors would like to thank the National Center for Protein Sciences at Peking University in Beijing, China, for assistance with the 4150 TapeStation System, Cell Imaging Multimode Reader (BioTek Cytation 5) and mass spectrometry; D. Liu and Q. Zhang for their help with mass spectrometry sample pretreatment and data analysis; the Center for Quantitative Biology at Peking University for assistance with the ImageXpress Micro 4 high-content imaging system and X. Li for her help; High Performance Computing Platform of the Center for Life Science (Peking University) for assistance with the analysis; and the Large-Scale Instrument Platform of State Key Laboratory of Natural and Biomimetic Drugs at Peking University for its advance and support in technology; Cystic Fibrosis Foundation for providing 16HBEge cells; and M. Zhang and Y. Ma for their discussion. This work was supported by the Ministry of Agriculture and Rural Affairs of China (NK2022010102 to C.Y.), the Natural Science Foundation of China (21825701 to C.Y. and 32200467 to Q.H.), the Ministry of Science and Technology of China (2023YFC3402200 to C.Y.), the Beijing Municipal Science and Technology Commission (Z231100002723005 to C.Y.) and the China Postdoctoral Science Foundation (2022M710222 to Q.H.).

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Author notes

  1. These authors contributed equally: Nan Luo, Qiang Huang, Liting Dong.

Authors and Affiliations

  1. State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China

    Nan Luo, Qiang Huang, Liting Dong, Jinghui Song, Hanxiao Sun, Hao Wu & Chengqi Yi

  2. Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China

    Liting Dong, Hao Wu & Chengqi Yi

  3. School of Life Sciences, Tsinghua University, Beijing, China

    Wenqing Liu

  4. Tsinghua-Peking Joint Center for Life Sciences, Tsinghua University, Beijing, China

    Wenqing Liu

  5. Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China

    Hao Wu

  6. Modit Therapeutics Beijing Limited, K115 Beijing ATLATL International Innovation Platform, Beijing, China

    Yuan Gao

  7. Department of Chemical Biology and Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, Beijing, China

    Chengqi Yi

  8. Beijing Advanced Center of RNA Biology (BEACON), Peking University, Beijing, China

    Chengqi Yi

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Contributions

C.Y. and N.L. conceived the project and designed the experiments. N.L., L.D. and Q.H. performed the experiments with the help of J.S., H.S. and Y.G. N.L. and L.D. designed and conducted all sample preparation for NGS. W.L. performed the bioinformatic analysis with the help of H.W. C.Y. supervised the project. C.Y., N.L., L.D. and Q.H. wrote the manuscript with contributions from all authors.

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Correspondence to Chengqi Yi.

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Supplementary Figs. 1–6.

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Supplementary Tables

Supplementary Table 1: Sequence of tRNA and primer. Supplementary Table 2: The composition of each version of the RESTART system (a) and the near-cognate tRNAs used in RESTART v3 and v3-mini (b). Supplementary Table 3: Primer information. Supplementary Table 4: Survey of human pathogenic nonsense mutations. Supplementary Table 5: list of detected off-target sites. Supplementary Table 6: Identification of the corresponding amino acid(s) incorporated at the off-target pseudouridine sites.

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Luo, N., Huang, Q., Dong, L. et al. Near-cognate tRNAs increase the efficiency and precision of pseudouridine-mediated readthrough of premature termination codons. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02165-8

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