1. Genetics and Genomics
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An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila

  1. David Li-Kroeger
  2. Oguz Kanca
  3. Pei-Tseng Lee
  4. Sierra Cowan
  5. Michael T Lee
  6. Manish Jaiswal
  7. Jose Louis Salazar
  8. Yuchun He
  9. Zhongyuan Zuo
  10. Hugo J Bellen  Is a corresponding author
  1. Baylor College of Medicine, United States
  2. Rice University, United States
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Cite this article as: eLife 2018;7:e38709 doi: 10.7554/eLife.38709

Abstract

We generated two new genetic tools to efficiently tag genes in Drosophila. The first, Double Header (DH) utilizes intronic MiMIC/CRIMIC insertions to generate artificial exons for GFP mediated protein trapping or T2A-GAL4 gene trapping in vivo based on Cre recombinase to avoid embryo injections. DH significantly increases integration efficiency compared to previous strategies and faithfully reports the expression pattern of genes and proteins. The second technique targets genes lacking coding introns using a two-step cassette exchange. First, we replace the endogenous gene with an excisable compact dominant marker using CRISPR making a null allele. Second, the insertion is replaced with a protein::tag cassette. This sequential manipulation allows the generation of numerous tagged alleles or insertion of other DNA fragments that facilitates multiple downstream applications. Both techniques allow precise gene manipulation and facilitate detection of gene expression, protein localization and assessment of protein function, as well as numerous other applications.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. The whole genome sequencing files are available on Zenodo (https://zenodo.org/record/1341241).

The following data sets were generated

Article and author information

Author details

  1. David Li-Kroeger

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.

  2. Oguz Kanca

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.

  3. Pei-Tseng Lee

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7501-7881

  4. Sierra Cowan

    Department of Biochemistry and Cell Biology, Rice University, Houston, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3530-9326

  5. Michael T Lee

    Department of Biochemistry and Cell Biology, Rice University, Houston, United States
    Competing interests
    No competing interests declared.

  6. Manish Jaiswal

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.

  7. Jose Louis Salazar

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.

  8. Yuchun He

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.

  9. Zhongyuan Zuo

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.

  10. Hugo J Bellen

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    For correspondence
    hbellen@bcm.edu
    Competing interests
    Hugo J Bellen, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5992-5989

Funding

National Institutes of Health (R01GM067858)

  • Hugo J Bellen

Howard Hughes Medical Institute

  • Hugo J Bellen

Robert A. and Renee E. Belfer Family Foundation

  • Hugo J Bellen

Huffington Foundation

  • Hugo J Bellen

National Institutes of Health (Training grant T32NS043124)

  • David Li-Kroeger

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

Reviewing Editor

  1. Yukiko M Yamashita, University of Michigan, United States

Publication history

  1. Received: May 28, 2018
  2. Accepted: July 25, 2018
  3. Accepted Manuscript published: August 9, 2018 (version 1)
  4. Version of Record published: August 16, 2018 (version 2)

Copyright

© 2018, Li-Kroeger 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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