Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach

  1. Ali Seleit  Is a corresponding author
  2. Alexander Aulehla
  3. Alexandre Paix  Is a corresponding author
  1. European Molecular Biology Laboratory, Germany

Abstract

The CRISPR/Cas9 system has been used to generate fluorescently labelled fusion proteins by homology directed repair in a variety of species. Despite its revolutionary success, there remains an urgent need for increased simplicity and efficiency of genome editing in research organisms. Here, we establish a simplified, highly efficient and precise strategy for CRISPR/Cas9 mediated endogenous protein tagging in medaka (Oryzias latipes). We use a cloning-free approach that relies on PCR amplified donor fragments containing the fluorescent reporter sequences flanked by short homology arms (30-40bp), a synthetic sgRNA and Cas9 mRNA. We generate eight novel knock-in lines with high efficiency of F0 targeting and germline transmission. Whole Genome Sequencing (WGS) results reveal single-copy integration events only at the targeted loci. We provide an initial characterization of these fusion-protein lines, significantly expanding the repertoire of genetic tools available in medaka. In particular, we show that the mScarlet-pcna line has the potential to serve as an organismal-wide label for proliferative zones and an endogenous cell cycle reporter.

Data availability

Sequencing data have been deposited in European Nucleotide Archive (ENA) under study number ERP127162. Accession numbers are: eGFP-cbx1b(1) ERS5796960 (SAMEA8109891), eGFP-cbx1b(2) ERS5796961 (SAMEA8109892), mScarlet-pcna ERS5796962 (SAMEA8109893) and mNeonGreen-myosinhc ERS5796963 (SAMEA8109894)

The following data sets were generated

Article and author information

Author details

  1. Ali Seleit

    Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
    For correspondence
    ali.seleit@embl.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8144-2286
  2. Alexander Aulehla

    Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3487-9239
  3. Alexandre Paix

    Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
    For correspondence
    alexandre.paix@embl.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8080-7546

Funding

H2020 European Research Council (866537)

  • Ali Seleit

EMBL interdisciplinary Postdoc (847543)

  • Ali Seleit

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

Ethics

Animal experimentation: Medaka (Oryzias latipes, Cab strain) (Iwamatsu, 2004, Naruse et al., 2004, Kasahara et al., 2007) were maintained as closed stocks in a fish facility built according to the European Union animal welfare standards and all animal experiments were performed in accordance with European Union animal welfare guidelines. Animal experimentation was approved by The EMBL Institutional Animal Care and Use Committee (IACUC) project code: 20/001_HD_AA. Fishes were maintained in a constant recirculating system at 27-28{degree sign}C with a 14hr light /10hr dark cycle.

Copyright

© 2021, Seleit 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

  • 7,166
    views
  • 746
    downloads
  • 27
    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. Ali Seleit
  2. Alexander Aulehla
  3. Alexandre Paix
(2021)
Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach
eLife 10:e75050.
https://doi.org/10.7554/eLife.75050

Share this article

https://doi.org/10.7554/eLife.75050

Further reading

    1. Developmental Biology
    2. Stem Cells and Regenerative Medicine
    Joshua G Medina-Feliciano, Griselle Valentín-Tirado ... José E Garcia-Arraras
    Research Article

    In holothurians, the regenerative process following evisceration involves the development of a ‘rudiment’ or ‘anlage’ at the injured end of the mesentery. This regenerating anlage plays a pivotal role in the formation of a new intestine. Despite its significance, our understanding of the molecular characteristics inherent to the constituent cells of this structure has remained limited. To address this gap, we employed state-of-the-art scRNA-seq and hybridization chain reaction fluorescent in situ hybridization analyses to discern the distinct cellular populations associated with the regeneration anlage. Through this approach, we successfully identified 13 distinct cell clusters. Among these, two clusters exhibit characteristics consistent with putative mesenchymal cells, while another four show features akin to coelomocyte cell populations. The remaining seven cell clusters collectively form a large group encompassing the coelomic epithelium of the regenerating anlage and mesentery. Within this large group of clusters, we recognized previously documented cell populations such as muscle precursors, neuroepithelial cells, and actively proliferating cells. Strikingly, our analysis provides data for identifying at least four other cellular populations that we define as the precursor cells of the growing anlage. Consequently, our findings strengthen the hypothesis that the coelomic epithelium of the anlage is a pluripotent tissue that gives rise to diverse cell types of the regenerating intestinal organ. Moreover, our results provide the initial view into the transcriptomic analysis of cell populations responsible for the amazing regenerative capabilities of echinoderms.

    1. Cell Biology
    2. Developmental Biology
    Yan Zhang, Hua Zhang
    Insight

    Long thought to have little relevance to ovarian physiology, the rete ovarii may have a role in follicular dynamics and reproductive health.