1. Genetics and Genomics

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
https://doi.org/10.7554/eLife.38709
Share this article
Cite this article
  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
  2. Download BibTeX
  3. Download .RIS

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.

Metrics

  • 6,599
    Page views
  • 958
    Downloads
  • 33
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

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

Categories and tags

Research organism

    1. Of interest

    Hedgehog regulation of epithelial cell state and morphogenesis in the larynx

    Janani Ramachandran, Weiqiang Zhou ... Steven A Vokes
  2. Further reading

Further reading

    1. Developmental Biology
    2. Genetics and Genomics

    Hedgehog regulation of epithelial cell state and morphogenesis in the larynx

    Janani Ramachandran, Weiqiang Zhou ... Steven A Vokes
    Research Article Updated

    The larynx enables speech while regulating swallowing and respiration. Larynx function hinges on the laryngeal epithelium which originates as part of the anterior foregut and undergoes extensive remodeling to separate from the esophagus and form vocal folds that interface with the adjacent trachea. Here we find that sonic hedgehog (SHH) is essential for epithelial integrity in the mouse larynx as well as the anterior foregut. During larynx-esophageal separation, low Shh expression marks specific domains of actively remodeling epithelium that undergo an epithelial-to-mesenchymal transition (EMT) characterized by the induction of N-Cadherin and movement of cells out of the epithelial layer. Consistent with a role for SHH signaling in regulating this process, Shh mutants undergo an abnormal EMT throughout the anterior foregut and larynx, marked by a cadherin switch, movement out of the epithelial layer and cell death. Unexpectedly, Shh mutant epithelial cells are replaced by a new population of FOXA2-negative cells that likely derive from adjacent pouch tissues and form a rudimentary epithelium. These findings have important implications for interpreting the etiology of HH-dependent birth defects within the foregut. We propose that SHH signaling has a default role in maintaining epithelial identity throughout the anterior foregut and that regionalized reductions in SHH trigger epithelial remodeling.

    1. Genetics and Genomics
    2. Plant Biology

    Evolutionary gain and loss of a plant pattern-recognition receptor for HAMP recognition

    Simon Snoeck, Bradley W Abramson ... Adam D Steinbrenner
    Research Article Updated

    As a first step in innate immunity, pattern recognition receptors (PRRs) recognize the distinct pathogen and herbivore-associated molecular patterns and mediate activation of immune responses, but specific steps in the evolution of new PRR sensing functions are not well understood. We employed comparative genomic and functional analyses to define evolutionary events leading to the sensing of the herbivore-associated peptide inceptin (In11) by the PRR inceptin receptor (INR) in legume plant species. Existing and de novo genome assemblies revealed that the presence of a functional INR gene corresponded with ability to respond to In11 across ~53 million years (my) of evolution. In11 recognition is unique to the clade of Phaseoloid legumes, and only a single clade of INR homologs from Phaseoloids was functional in a heterologous model. The syntenic loci of several non-Phaseoloid outgroup species nonetheless contain non-functional INR-like homologs, suggesting that an ancestral gene insertion event and diversification preceded the evolution of a specific INR receptor function ~28 my ago. Chimeric and ancestrally reconstructed receptors indicated that 16 amino acid differences in the C1 leucine-rich repeat domain and C2 intervening motif mediate gain of In11 recognition. Thus, high PRR diversity was likely followed by a small number of mutations to expand innate immune recognition to a novel peptide elicitor. Analysis of INR evolution provides a model for functional diversification of other germline-encoded PRRs.

    1. Chromosomes and Gene Expression
    2. Genetics and Genomics

    Drosophila SUMM4 complex couples insulator function and DNA replication control

    Evgeniya N Andreyeva, Alexander V Emelyanov ... Dmitry V Fyodorov
    Research Article

    Asynchronous replication of chromosome domains during S phase is essential for eukaryotic genome function, but the mechanisms establishing which domains replicate early versus late in different cell types remain incompletely understood. Intercalary heterochromatin domains replicate very late in both diploid chromosomes of dividing cells and in endoreplicating polytene chromosomes where they are also underrelicated. Drosophila SNF2-related factor SUUR imparts locus-specific underreplication of polytene chromosomes. SUUR negatively regulates DNA replication fork progression; however, its mechanism of action remains obscure. Here we developed a novel method termed MS-Enabled Rapid protein Complex Identification (MERCI) to isolate a stable stoichiometric native complex SUMM4 that comprises SUUR and a chromatin boundary protein Mod(Mdg4)-67.2. Mod(Mdg4) stimulates SUUR ATPase activity and is required for a normal spatiotemporal distribution of SUUR in vivo. SUUR and Mod(Mdg4)-67.2 together mediate the activities of gypsy insulator that prevent certain enhancer-promoter interactions and establish euchromatin-heterochromatin barriers in the genome. Furthermore, SuUR or mod(mdg4) mutations reverse underreplication of intercalary heterochromatin. Thus, SUMM4 can impart late replication of intercalary heterochromatin by attenuating the progression of replication forks through euchromatin/heterochromatin boundaries. Our findings implicate a SNF2 family ATP-dependent motor protein SUUR in the insulator function, reveal that DNA replication can be delayed by a chromatin barrier and uncover a critical role for architectural proteins in replication control. They suggest a mechanism for the establishment of late replication that does not depend on an asynchronous firing of late replication origins.