An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila

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.

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.

Metrics

  • 7,722
    views
  • 1,082
    downloads
  • 68
    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. 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
(2018)
An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila
eLife 7:e38709.
https://doi.org/10.7554/eLife.38709

Share this article

https://doi.org/10.7554/eLife.38709

Further reading

    1. Genetics and Genomics
    Yi Li, Long Gong ... Shangbang Gao
    Research Article

    Resistance to anthelmintics, particularly the macrocyclic lactone ivermectin (IVM), presents a substantial global challenge for parasite control. We found that the functional loss of an evolutionarily conserved E3 ubiquitin ligase, UBR-1, leads to IVM resistance in Caenorhabditis elegans. Multiple IVM-inhibiting activities, including viability, body size, pharyngeal pumping, and locomotion, were significantly ameliorated in various ubr-1 mutants. Interestingly, exogenous application of glutamate induces IVM resistance in wild-type animals. The sensitivity of all IVM-affected phenotypes of ubr-1 is restored by eliminating proteins associated with glutamate metabolism or signaling: GOT-1, a transaminase that converts aspartate to glutamate, and EAT-4, a vesicular glutamate transporter. We demonstrated that IVM-targeted GluCls (glutamate-gated chloride channels) are downregulated and that the IVM-mediated inhibition of serotonin-activated pharynx Ca2+ activity is diminished in ubr-1. Additionally, enhancing glutamate uptake in ubr-1 mutants through ceftriaxone completely restored their IVM sensitivity. Therefore, UBR-1 deficiency-mediated aberrant glutamate signaling leads to ivermectin resistance in C. elegans.

    1. Genetics and Genomics
    Minsoo Noh, Xiangguo Che ... Sihoon Lee
    Research Article

    Osteoporosis, characterized by reduced bone density and strength, increases fracture risk, pain, and limits mobility. Established therapies of parathyroid hormone (PTH) analogs effectively promote bone formation and reduce fractures in severe osteoporosis, but their use is limited by potential adverse effects. In the pursuit of safer osteoporosis treatments, we investigated R25CPTH, a PTH variant wherein the native arginine at position 25 is substituted by cysteine. These studies were prompted by our finding of high bone mineral density in a hypoparathyroidism patient with the R25C homozygous mutation, and we explored its effects on PTH type-1 receptor (PTH1R) signaling in cells and bone metabolism in mice. Our findings indicate that R25CPTH(1–84) forms dimers both intracellularly and extracellularly, and the synthetic dimeric peptide, R25CPTH(1–34), exhibits altered activity in PTH1R-mediated cyclic AMP (cAMP) response. Upon a single injection in mice, dimeric R25CPTH(1–34) induced acute calcemic and phosphaturic responses comparable to PTH(1–34). Furthermore, repeated daily injections increased calvarial bone thickness in intact mice and improved trabecular and cortical bone parameters in ovariectomized (OVX) mice, akin to PTH(1–34). The overall results reveal a capacity of a dimeric PTH peptide ligand to activate the PTH1R in vitro and in vivo as PTH, suggesting a potential path of therapeutic PTH analog development.