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Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure

Author

Listed:
  • Xiaosa Li

    (Shanghai Jiao Tong University School of Medicine
    Shanghai Key Laboratory of Fundus Diseases
    ShanghaiTech University)

  • Lina Zhou

    (ShanghaiTech University
    Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences
    University of Chinese Academy of Sciences
    Shanghai Clinical Research and Trial Center)

  • Bao-Qing Gao

    (University of Chinese Academy of Sciences, Chinese Academy of Sciences)

  • Guangye Li

    (ShanghaiTech University
    Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences
    University of Chinese Academy of Sciences)

  • Xiao Wang

    (ShanghaiTech University
    Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences
    University of Chinese Academy of Sciences)

  • Ying Wang

    (University of Chinese Academy of Sciences, Chinese Academy of Sciences)

  • Jia Wei

    (University of Chinese Academy of Sciences, Chinese Academy of Sciences)

  • Wenyan Han

    (ShanghaiTech University
    Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences
    University of Chinese Academy of Sciences)

  • Zixian Wang

    (ShanghaiTech University)

  • Jifang Li

    (ShanghaiTech University
    Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences
    University of Chinese Academy of Sciences)

  • Runze Gao

    (ShanghaiTech University
    Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences
    University of Chinese Academy of Sciences)

  • Junjie Zhu

    (ShanghaiTech University
    Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences
    University of Chinese Academy of Sciences)

  • Wenchao Xu

    (ShanghaiTech University
    Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences
    University of Chinese Academy of Sciences)

  • Jing Wu

    (ShanghaiTech University)

  • Bei Yang

    (Shanghai Clinical Research and Trial Center
    ShanghaiTech University)

  • Xiaodong Sun

    (Shanghai Jiao Tong University School of Medicine
    Shanghai Key Laboratory of Fundus Diseases)

  • Li Yang

    (Fudan University and Shanghai Key Laboratory of Medical Epigenetics, International Laboratory of Medical Epigenetics and Metabolism, Ministry of Science and Technology, Institutes of Biomedical Sciences, Fudan University)

  • Jia Chen

    (ShanghaiTech University
    Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences
    Shanghai Clinical Research and Trial Center)

Abstract

Prime editor (PE), which is developed by combining Cas9 nickase and an engineered reverse transcriptase, can mediate all twelve types of base substitutions and small insertions or deletions in living cells but its efficiency remains low. Here, we develop spegRNA by introducing same-sense mutations at proper positions in the reverse-transcription template of pegRNA to increase PE’s base-editing efficiency up-to 4,976-fold (on-average 353-fold). We also develop apegRNA by altering the pegRNA secondary structure to increase PE’s indel-editing efficiency up-to 10.6-fold (on-average 2.77-fold). The spegRNA and apegRNA can be combined to further enhance editing efficiency. When spegRNA and apegRNA are used in PE3 and PE5 systems, the efficiencies of sPE3, aPE3, sPE5 and aPE5 systems are all enhanced significantly. The strategies developed in this study realize highly efficient prime editing at certain previously uneditable sites.

Suggested Citation

  • Xiaosa Li & Lina Zhou & Bao-Qing Gao & Guangye Li & Xiao Wang & Ying Wang & Jia Wei & Wenyan Han & Zixian Wang & Jifang Li & Runze Gao & Junjie Zhu & Wenchao Xu & Jing Wu & Bei Yang & Xiaodong Sun & L, 2022. "Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure," Nature Communications, Nature, vol. 13(1), pages 1-9, December.
  • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-29339-9
    DOI: 10.1038/s41467-022-29339-9
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    References listed on IDEAS

    as
    1. Pengpeng Liu & Shun-Qing Liang & Chunwei Zheng & Esther Mintzer & Yan G. Zhao & Karthikeyan Ponnienselvan & Aamir Mir & Erik J. Sontheimer & Guangping Gao & Terence R. Flotte & Scot A. Wolfe & Wen Xue, 2021. "Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice," Nature Communications, Nature, vol. 12(1), pages 1-13, December.
    2. Jonathan Y. Hsu & Julian Grünewald & Regan Szalay & Justine Shih & Andrew V. Anzalone & Kin Chung Lam & Max W. Shen & Karl Petri & David R. Liu & J. Keith Joung & Luca Pinello, 2021. "PrimeDesign software for rapid and simplified design of prime editing guide RNAs," Nature Communications, Nature, vol. 12(1), pages 1-6, December.
    3. Andrew V. Anzalone & Peyton B. Randolph & Jessie R. Davis & Alexander A. Sousa & Luke W. Koblan & Jonathan M. Levy & Peter J. Chen & Christopher Wilson & Gregory A. Newby & Aditya Raguram & David R. L, 2019. "Search-and-replace genome editing without double-strand breaks or donor DNA," Nature, Nature, vol. 576(7785), pages 149-157, December.
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    Cited by:

    1. Jianli Tao & Daniel E. Bauer & Roberto Chiarle, 2023. "Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing," Nature Communications, Nature, vol. 14(1), pages 1-16, December.
    2. Jaesuk Lee & Kayeong Lim & Annie Kim & Young Geun Mok & Eugene Chung & Sung-Ik Cho & Ji Min Lee & Jin-Soo Kim, 2023. "Prime editing with genuine Cas9 nickases minimizes unwanted indels," Nature Communications, Nature, vol. 14(1), pages 1-11, December.

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