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.

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. 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.

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.

Metrics

  • 15,432
    views
  • 1,905
    downloads
  • 131
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Fillip Port
  2. Claudia Strein
  3. Mona Stricker
  4. Benedikt Rauscher
  5. Florian Heigwer
  6. Jun Zhou
  7. Celine Beyersdörffer
  8. Jana Frei
  9. Amy Hess
  10. Katharina Kern
  11. Laura Lange
  12. Nora Langner
  13. Roberta Malamud
  14. Bojana Pavlovic
  15. Kristin Rädecke
  16. Lukas Schmitt
  17. Lukas Voos
  18. Erica Valentini
  19. Michael Boutros
(2020)
A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila
eLife 9:e53865.
https://doi.org/10.7554/eLife.53865

Share this article

https://doi.org/10.7554/eLife.53865

Further reading

    1. Developmental Biology
    Bin Zhu, Rui Wei ... Pei Liang
    Research Article

    Wing dimorphism is a common phenomenon that plays key roles in the environmental adaptation of aphid; however, the signal transduction in response to environmental cues and the regulation mechanism related to this event remain unknown. Adenosine (A) to inosine (I) RNA editing is a post-transcriptional modification that extends transcriptome variety without altering the genome, playing essential roles in numerous biological and physiological processes. Here, we present a chromosome-level genome assembly of the rose-grain aphid Metopolophium dirhodum by using PacBio long HiFi reads and Hi-C technology. The final genome assembly for M. dirhodum is 447.8 Mb, with 98.50% of the assembled sequences anchored to nine chromosomes. The contig and scaffold N50 values are 7.82 and 37.54 Mb, respectively. A total of 18,003 protein-coding genes were predicted, of which 92.05% were functionally annotated. In addition, 11,678 A-to-I RNA-editing sites were systematically identified based on this assembled M. dirhodum genome, and two synonymous A-to-I RNA-editing sites on CYP18A1 were closely associated with transgenerational wing dimorphism induced by crowding. One of these A-to-I RNA-editing sites may prevent the binding of miR-3036-5p to CYP18A1, thus elevating CYP18A1 expression, decreasing 20E titer, and finally regulating the wing dimorphism of offspring. Meanwhile, crowding can also inhibit miR-3036-5p expression and further increase CYP18A1 abundance, resulting in winged offspring. These findings support that A-to-I RNA editing is a dynamic mechanism in the regulation of transgenerational wing dimorphism in aphids and would advance our understanding of the roles of RNA editing in environmental adaptability and phenotypic plasticity.

    1. Developmental Biology
    Hanee Lee, Junsu Kang ... Junho Lee
    Research Article

    The evolutionarily conserved Hippo (Hpo) pathway has been shown to impact early development and tumorigenesis by governing cell proliferation and apoptosis. However, its post-developmental roles are relatively unexplored. Here, we demonstrate its roles in post-mitotic cells by showing that defective Hpo signaling accelerates age-associated structural and functional decline of neurons in Caenorhabditis elegans. Loss of wts-1/LATS, the core kinase of the Hpo pathway, resulted in premature deformation of touch neurons and impaired touch responses in a yap-1/YAP-dependent manner, the downstream transcriptional co-activator of LATS. Decreased movement as well as microtubule destabilization by treatment with colchicine or disruption of microtubule-stabilizing genes alleviated the neuronal deformation of wts-1 mutants. Colchicine exerted neuroprotective effects even during normal aging. In addition, the deficiency of a microtubule-severing enzyme spas-1 also led to precocious structural deformation. These results consistently suggest that hyper-stabilized microtubules in both wts-1-deficient neurons and normally aged neurons are detrimental to the maintenance of neuronal structural integrity. In summary, Hpo pathway governs the structural and functional maintenance of differentiated neurons by modulating microtubule stability, raising the possibility that the microtubule stability of fully developed neurons could be a promising target to delay neuronal aging. Our study provides potential therapeutic approaches to combat age- or disease-related neurodegeneration.