1. Genetics and Genomics
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One-step efficient generation of dual-function conditional knockout and geno-tagging alleles in zebrafish

  1. Wenyuan Li
  2. Yage Zhang
  3. Bingzhou Han
  4. Lianyan Li
  5. Muhang Li
  6. Xiaochan Lu
  7. Cheng Chen
  8. Mengjia Lu
  9. Yujie Zhang
  10. Xuefeng Jia
  11. Zuoyan zhu
  12. Xiangjun Tong
  13. Bo Zhang  Is a corresponding author
  1. Peking University, China
  2. Peking University Shenzhen Graduate School, China
  3. Gcrispr (Tianjin) Genetic Technology, China
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Cite this article as: eLife 2019;8:e48081 doi: 10.7554/eLife.48081

Abstract

CRISPR/Cas systems are widely used to knockout genes by inducing indel mutations, which are prone to genetic compensation. Complex genome modifications such as knockin (KI) might bypass compensation, though difficult to practice due to low efficiency. Moreover, no 'two-in-one' KI strategy combining conditional knockout (CKO) with fluorescent gene-labeling or further allele-labeling has been reported. Here, we developed a dual-cassette-donor strategy and achieved one-step and efficient generation of dual-function KI alleles at tbx5a and kctd10 loci in zebrafish via targeted insertion. These alleles display fluorescent gene-tagging and CKO effects before and after Cre induction, respectively. By introducing a second fluorescent reporter, geno-tagging effects were achieved at tbx5a and sox10 loci, exhibiting CKO coupled with fluorescent reporter switch upon Cre induction, enabling tracing of three distinct genotypes. We found that LiCl purification of gRNA is critical for highly efficient KI, and preselection of founders allows the efficient germline recovery of KI events.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Wenyuan Li

    College of Life Sciences, Peking University, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  2. Yage Zhang

    College of Life Sciences, Peking University, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  3. Bingzhou Han

    College of Life Sciences, Peking University, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  4. Lianyan Li

    College of Life Sciences, Peking University, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  5. Muhang Li

    College of Life Sciences, Peking University, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  6. Xiaochan Lu

    School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, China
    Competing interests
    The authors declare that no competing interests exist.
  7. Cheng Chen

    School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, China
    Competing interests
    The authors declare that no competing interests exist.
  8. Mengjia Lu

    School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, China
    Competing interests
    The authors declare that no competing interests exist.
  9. Yujie Zhang

    College of Life Sciences, Peking University, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5038-1487
  10. Xuefeng Jia

    Gcrispr (Tianjin) Genetic Technology, Tianjin, China
    Competing interests
    The authors declare that no competing interests exist.
  11. Zuoyan zhu

    College of Life Sciences, Peking University, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  12. Xiangjun Tong

    College of Life Sciences, Peking University, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  13. Bo Zhang

    College of Life Sciences, Peking University, Beijing, China
    For correspondence
    bzhang@pku.edu.cn
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6436-5629

Funding

National Key Research and Development Program of China (2018YFA0801000)

  • Bo Zhang

National Key Research and Development Program of China (2016YFA0100500)

  • Bo Zhang

National Key Basic Research Program of China (2015CB942803)

  • Bo Zhang

National Natural Science Foundation of China (31671500)

  • Bo Zhang

National Natural Science Foundation of China (31871458)

  • Bo Zhang

National Natural Science Foundation of China (81371264)

  • Bo Zhang

PKU Qidong-SLS Innovation Fund

  • Bo Zhang

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

Ethics

Animal experimentation: All animal experiments were approved by Institutional Animal Care and Use Committee (IACUC) of Peking University. The reference from IACUC of Peking University is LSC-ZhangB-2.

Reviewing Editor

  1. Darius Balciunas, Temple University, United States

Publication history

  1. Received: April 30, 2019
  2. Accepted: October 30, 2019
  3. Accepted Manuscript published: October 30, 2019 (version 1)
  4. Version of Record published: November 11, 2019 (version 2)

Copyright

© 2019, Li 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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