A high-throughput small molecule screen identifies farrerol as a potentiator of CRISPR/Cas9-mediated genome editing

  1. Weina Zhang
  2. Yu Chen
  3. Jiaqing Yang
  4. Jing Zhang
  5. Jiayu Yu
  6. Mengting Wang
  7. Xiaodong Zhao
  8. Ke Wei
  9. Xiaoping Wan
  10. Xiaojun Xu
  11. Ying Jiang
  12. Jiayu Chen  Is a corresponding author
  13. Shaorong Gao  Is a corresponding author
  14. Zhiyong Mao  Is a corresponding author
  1. Tongji University, China
  2. China Pharmaceutical University, China

Abstract

Directly modulating the choice between homologous recombination (HR) and non-homologous end joining (NHEJ) - two independent pathways for repairing DNA double-strand breaks (DSBs) - has the potential to improve the efficiency of gene targeting by CRISPR/Cas9. Here, we have developed a rapid and easy-to-score screening approach for identifying small molecules that affect the choice between the two DSB repair pathways. Using this tool, we identified a small molecule, farrerol, that promotes HR but does not affect NHEJ. Further mechanistic studies indicate that farrerol functions through stimulating the recruitment of RAD51 to DSB sites. Importantly, we demonstrated that farrerol effectively promotes precise targeted integration in human cells, mouse cells and mouse embryos at multiple genomic loci. In addition, treating cells with farrerol did not have any obvious negative effect on genomic stability. Moreover, farrerol significantly improved the knock-in efficiency in blastocysts, and the subsequently generated knock-in mice retained the capacity for germline transmission.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figure 1, 2, 3, 4, 5, 6 and figure supplements contained within 'Source data files'. Primer sequences named 'Table 1-source data 1' are also included as 'Source data files'.

Article and author information

Author details

  1. Weina Zhang

    School of Life Science and Technology, Tongji University, Shanghai, China
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2852-4079
  2. Yu Chen

    School of life sciences and technology, Tongji University, Shanghai, China
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9661-3914
  3. Jiaqing Yang

    School of Life Science and Technology, Tongji University, Shanghai, China
    Competing interests
    The authors declare that no competing interests exist.
  4. Jing Zhang

    School of Life Science and Technology, Tongji University, Shanghai, China
    Competing interests
    The authors declare that no competing interests exist.
  5. Jiayu Yu

    School of Life Science and Technology, Tongji University, Shanghai, China
    Competing interests
    The authors declare that no competing interests exist.
  6. Mengting Wang

    School of Life Science and Technology, Tongji University, Shanghai, China
    Competing interests
    The authors declare that no competing interests exist.
  7. Xiaodong Zhao

    School of Life Science and Technology, Tongji University, Shanghai, China
    Competing interests
    The authors declare that no competing interests exist.
  8. Ke Wei

    School of Life Science and Technology, Tongji University, Shanghai, China
    Competing interests
    The authors declare that no competing interests exist.
  9. Xiaoping Wan

    School of Life Science and Technology, Tongji University, Shanghai, China
    Competing interests
    The authors declare that no competing interests exist.
  10. Xiaojun Xu

    State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, China
    Competing interests
    The authors declare that no competing interests exist.
  11. Ying Jiang

    School of Life Science and Technology, Tongji University, Shanghai, China
    Competing interests
    The authors declare that no competing interests exist.
  12. Jiayu Chen

    School of Life Science and Technology, Tongji University, Shanghai, China
    For correspondence
    chenjiayu@tongji.edu.cn
    Competing interests
    The authors declare that no competing interests exist.
  13. Shaorong Gao

    School of Life Sciences and Technology, Tongji University, Shanghai, China
    For correspondence
    gaoshaorong@tongji.edu.cn
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1041-3928
  14. Zhiyong Mao

    School of Life Science and Technology, Tongji University, Shanghai, China
    For correspondence
    zhiyong_mao@tongji.edu.cn
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5298-1918

Funding

Chinese National Program on Key Basic Research Project (2018YFC2000100)

  • Zhiyong Mao

the key project of the Science and Technology of Shanghai Municipality (19JC1415300)

  • Shaorong Gao

the Shanghai Rising-Star Program (19QA1409600)

  • Jiayu Chen

the Shanghai Municipal Medical and Health Discipline Construction Projects (2017ZZ02015)

  • Xiaoping Wan

the Young Elite Scientist Sponsorship Program by CAST (2018QNRC001)

  • Jiayu Chen

Chinese National Program on Key Basic Research Project (2017YFA010330)

  • Ying Jiang

Chinese National Program on Key Basic Research Project (2016YFA0100400)

  • Shaorong Gao

the National Science Foundation of China (31871438)

  • Zhiyong Mao

the National Science Foundation of China (81972457)

  • Ying Jiang

the National Science Foundation of China (31721003)

  • Shaorong Gao

the National Science Foundation of China (31871446)

  • Jiayu Chen

the Fundamental Research Funds for the Central Universities, Program of Shanghai Academic Research Leader (19XD1403000)

  • Zhiyong Mao

Shuguang Program" of Shanghai Education Development Foundation and Shanghai Municipal Education Commission" (19SG18)

  • Zhiyong Mao

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: The specific-pathogen-free-grade mice, including C57BL/6n, ICR, and BDF1 mice, were housed in the animal facility at Tongji University. All the mice had free access to food and water. All the experiments were performed in accordance with the University of Health Guide for the Care and Use of Laboratory Animals, and were approved by the Biological Research Ethics Committee of Tongji University, and the approved protocol number was TJLAC-019-095.

Reviewing Editor

  1. Wolf-Dietrich Heyer, University of California, Davis, United States

Publication history

  1. Received: February 13, 2020
  2. Accepted: July 8, 2020
  3. Accepted Manuscript published: July 9, 2020 (version 1)
  4. Version of Record published: July 24, 2020 (version 2)

Copyright

© 2020, Zhang et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Weina Zhang
  2. Yu Chen
  3. Jiaqing Yang
  4. Jing Zhang
  5. Jiayu Yu
  6. Mengting Wang
  7. Xiaodong Zhao
  8. Ke Wei
  9. Xiaoping Wan
  10. Xiaojun Xu
  11. Ying Jiang
  12. Jiayu Chen
  13. Shaorong Gao
  14. Zhiyong Mao
(2020)
A high-throughput small molecule screen identifies farrerol as a potentiator of CRISPR/Cas9-mediated genome editing
eLife 9:e56008.
https://doi.org/10.7554/eLife.56008

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