An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR mediated homologous recombination

  1. Oguz Kanca  Is a corresponding author
  2. Jonathan Zirin
  3. Yanhui Hu
  4. Burak Tepe
  5. Debdeep Dutta
  6. Wen-Wen Lin
  7. Liwen Ma
  8. Ming Ge
  9. Zhongyuan Zuo
  10. Lu-Ping Liu
  11. Robert W Levis
  12. Norbert Perrimon
  13. Hugo J Bellen  Is a corresponding author
  1. Baylor College of Medicine, United States
  2. Harvard Medical School, United States
  3. Carnegie Institution for Science, United States

Abstract

Previously, we described a large collection of Drosophila strains that each carry an artificial exon containing a T2AGAL4 cassette inserted in an intron of a target gene based on CRISPR-mediated homologous recombination (Lee et al., 2018). These alleles permit numerous applications and have proven to be very useful. Initially, the homologous recombination-based donor constructs had long homology arms (>500 bps) to promote precise integration of large constructs (>5kb). Recently, we showed that in vivo linearization of the donor constructs enables insertion of large artificial exons in introns using short homology arms (100-200 bps) (Kanca et al., 2019a). Shorter homology arms make it feasible to commercially synthesize homology donors and minimize the cloning steps for donor construct generation. Unfortunately, about 58% of Drosophila genes lack a suitable coding intron for integration of artificial exons in all of the annotated isoforms. Here, we report the development of new set of constructs that allow the replacement of the coding region of genes that lack suitable introns with a KozakGAL4 cassette, generating a knock-out/knock-in allele that expresses GAL4 similarly as the targeted gene. We also developed custom vector backbones to further facilitate and improve transgenesis. Synthesis of homology donor constructs in custom plasmid backbones that contain the target gene sgRNA obviates the need to inject a separate sgRNA plasmid and significantly increases the transgenesis efficiency. These upgrades will enable the targeting of nearly every fly gene, regardless of exon-intron structure, with a 70-80% success rate.

Data availability

Source data for the graphs in Supplementary table 1 is included above the graphs in Supplementary table 1. The list of all the generated alleles and the method used can be found in Supplementary table 2.

The following previously published data sets were used

Article and author information

Author details

  1. Oguz Kanca

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    For correspondence
    kanca@bcm.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5438-0879
  2. Jonathan Zirin

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Yanhui Hu

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Burak Tepe

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Debdeep Dutta

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Wen-Wen Lin

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Liwen Ma

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Ming Ge

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Zhongyuan Zuo

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Lu-Ping Liu

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Robert W Levis

    Department of Embryology, Carnegie Institution for Science, Baltimore, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3453-2390
  12. Norbert Perrimon

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7542-472X
  13. Hugo J Bellen

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    For correspondence
    hbellen@bcm.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5992-5989

Funding

National Institute of General Medical Sciences (R01GM067858)

  • Hugo J Bellen

Office of Research Infrastructure Programs, National Institutes of Health (R24OD031447)

  • Oguz Kanca
  • Hugo J Bellen

Office of Research Infrastructure Programs, National Institutes of Health (R24OD031447)

  • Hugo J Bellen

National Institute of Neurological Disorders and Stroke (U54NS093793)

  • Hugo J Bellen

Huffington Foundation

  • Hugo J Bellen

National Institute of General Medical Sciences (GM067761)

  • Jonathan Zirin
  • Yanhui Hu
  • Norbert Perrimon

National Institute of General Medical Sciences (GM084947)

  • Jonathan Zirin
  • Yanhui Hu
  • Norbert Perrimon

Howard Hughes Medical Institute

  • Norbert Perrimon

Carnegie Institution for Science

  • Robert W Levis

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

Reviewing Editor

  1. K VijayRaghavan, National Centre for Biological Sciences, Tata Institute of Fundamental Research, India

Publication history

  1. Received: December 7, 2021
  2. Preprint posted: December 24, 2021 (view preprint)
  3. Accepted: June 19, 2022
  4. Accepted Manuscript published: June 20, 2022 (version 1)
  5. Version of Record published: June 28, 2022 (version 2)

Copyright

© 2022, Kanca 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. Oguz Kanca
  2. Jonathan Zirin
  3. Yanhui Hu
  4. Burak Tepe
  5. Debdeep Dutta
  6. Wen-Wen Lin
  7. Liwen Ma
  8. Ming Ge
  9. Zhongyuan Zuo
  10. Lu-Ping Liu
  11. Robert W Levis
  12. Norbert Perrimon
  13. Hugo J Bellen
(2022)
An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR mediated homologous recombination
eLife 11:e76077.
https://doi.org/10.7554/eLife.76077

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