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A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila

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Cite this article as: eLife 2020;9:e53865 doi: 10.7554/eLife.53865

Abstract

Genetic screens are powerful tools for the functional annotation of genomes. In the context of multicellular organisms, interrogation of gene function is greatly facilitated by methods that allow spatial and temporal control of gene abrogation. Here, we describe a large-scale transgenic short guide (sg) RNA library for efficient CRISPR-based disruption of specific target genes in a constitutive or conditional manner. The library consists currently of more than 2600 plasmids and 1700 fly lines with a focus on targeting kinases, phosphatases and transcription factors, each expressing two sgRNAs under control of the Gal4/UAS system. We show that conditional CRISPR mutagenesis is robust across many target genes and can be efficiently employed in various somatic tissues, as well as the germline. In order to prevent artefacts commonly associated with excessive amounts of Cas9 protein, we have developed a series of novel UAS-Cas9 transgenes, which allow fine tuning of Cas9 expression to achieve high gene editing activity without detectable toxicity. Functional assays, as well as direct sequencing of genomic sgRNA target sites, indicates that the vast majority of transgenic sgRNA lines mediate efficient gene disruption. Furthermore, we conducted the so far largest fully transgenic CRISPR screen in any metazoan organism, which further supported the high efficiency and accuracy of our library and revealed many so far uncharacterized genes essential for development.

Article and author information

Author details

  1. Fillip Port

    Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Heidelberg University, Heidelberg, Germany
    For correspondence
    f.port@dkfz.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5157-4835
  2. Claudia Strein

    Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Mona Stricker

    Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Benedikt Rauscher

    Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Florian Heigwer

    Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8230-1485
  6. Jun Zhou

    Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2101-9304
  7. Celine Beyersdörffer

    Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  8. Jana Frei

    Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  9. Amy Hess

    Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  10. Katharina Kern

    Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  11. Laura Lange

    Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  12. Nora Langner

    Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  13. Roberta Malamud

    Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  14. Bojana Pavlovic

    Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  15. Kristin Rädecke

    Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  16. Lukas Schmitt

    Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  17. Lukas Voos

    Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  18. Erica Valentini

    Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Heidelberg University, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  19. Michael Boutros

    Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ) and Heidelberg University, Heidelberg, Germany
    For correspondence
    m.boutros@dkfz.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9458-817X

Funding

Deutsche Forschungsgemeinschaft (TRR186)

  • Fillip Port
  • Bojana Pavlovic
  • Michael Boutros

European Research Council (Decode)

  • Michael Boutros

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

Reviewing Editor

  1. Michael B Eisen, HHMI, University of California, Berkeley, United States

Publication history

  1. Received: November 22, 2019
  2. Accepted: February 1, 2020
  3. Accepted Manuscript published: February 13, 2020 (version 1)
  4. Version of Record published: March 9, 2020 (version 2)

Copyright

© 2020, Port 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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