Cre/lox regulated conditional rescue and inactivation with zebrafish UFlip alleles generated by CRISPR-Cas9 targeted integration

Abstract

The ability to regulate gene activity spatially and temporally is essential to investigate cell type-specific gene function during development and in postembryonic processes and disease models. The Cre/lox system has been widely used for performing cell and tissue-specific conditional analysis of gene function in zebrafish. However, simple and efficient methods for isolation of stable, Cre/lox regulated zebrafish alleles are lacking. Here we applied our GeneWeld CRISPR-Cas9 targeted integration strategy to generate floxed alleles that provide robust conditional inactivation and rescue. A universal targeting vector, UFlip, with sites for cloning short homology arms flanking a floxed 2A-mRFP gene trap, was integrated into an intron in rbbp4 and rb1. rbbp4off and rb1off integration alleles resulted in strong mRFP expression, >99% reduction of endogenous gene expression, and recapitulated known indel loss of function phenotypes. Introduction of Cre led to stable inversion of the floxed cassette, loss of mRFP expression, and phenotypic rescue. rbbp4on and rb1on integration alleles did not cause phenotypes in combination with a loss of function mutation. Addition of Cre led to conditional inactivation by stable inversion of the cassette, gene trapping and mRFP expression, and the expected mutant phenotype. Neural progenitor Cre drivers were used for conditional inactivation and phenotypic rescue to showcase how this approach can be used in specific cell populations. Together these results validate a simplified approach for efficient isolation of Cre/lox responsive conditional alleles in zebrafish. Our strategy provides a new toolkit for generating genetic mosaics and represents a significant advance in zebrafish genetics.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Table 2, Figure 4, Figure 4 - figure supplement 1, Figure 5, Figure 5 - figure supplement 2, Figure 6, Figure 6 - figure supplement 2, Figure 7, Figure 7 - figure supplement 1, Figure 7 - figure supplement 2, Figure 8, Figure 9

Article and author information

Author details

  1. Fang Liu

    Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States
    Competing interests
    No competing interests declared.
  2. Sekhar Kambakam

    Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3331-3754
  3. Maira P Almeida

    Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States
    Competing interests
    No competing interests declared.
  4. Zhitao Ming

    Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States
    Competing interests
    No competing interests declared.
  5. Jordan M Welker

    Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States
    Competing interests
    No competing interests declared.
  6. Wesley A Wierson

    Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States
    Competing interests
    Wesley A Wierson, WAW has competing interests with LifEngine and LifEngine Animal Health.
  7. Laura E Schultz-Rogers

    Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States
    Competing interests
    No competing interests declared.
  8. Stephen C Ekker

    Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, United States
    Competing interests
    Stephen C Ekker, Reviewing editor, eLife. Has competing interests with LifEngine and LifEngine Animal Health.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0726-4212
  9. Karl J Clark

    Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, United States
    Competing interests
    Karl J Clark, has competing interests with Recombinetics Inc., LifEngine and LifEngine Animal Health.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9637-0967
  10. Jeffrey J Essner

    Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States
    Competing interests
    Jeffrey J Essner, has competing interests with Recombinetics Inc., Immusoft Inc., LifEngine and LifEngine Animal Health.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8816-3848
  11. Maura McGrail

    Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States
    For correspondence
    mmcgrail@iastate.edu
    Competing interests
    Maura McGrail, has competing interests with Recombinetics Inc., Immusoft Inc., LifEngine and LifEngine Animal Health.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9308-6189

Funding

NIH Office of the Director (R24 OD 020166)

  • Stephen C Ekker
  • Karl J Clark
  • Jeffrey J Essner
  • Maura McGrail

CNPq Brazilian National Council for Scientific and Technological Development

  • Maira P Almeida

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

Ethics

Animal experimentation: Use of zebrafish for research in this study was performed according to the Guidelines for Ethical Conduct in the Care and Use of Animals (APA, 1986), and carried out in accordance with Iowa State University Animal Care and Use Committee IACUC-18-279 and IACUC-20-058 approved protocols. All methods involving zebrafish were in compliance with the American Veterinary Medical Association (2020), ARRIVE (Percie du Sert et al., 2020) and NIH guidelines for the humane use of animals in research.

Copyright

© 2022, Liu 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

  • 4,848
    views
  • 888
    downloads
  • 19
    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. Fang Liu
  2. Sekhar Kambakam
  3. Maira P Almeida
  4. Zhitao Ming
  5. Jordan M Welker
  6. Wesley A Wierson
  7. Laura E Schultz-Rogers
  8. Stephen C Ekker
  9. Karl J Clark
  10. Jeffrey J Essner
  11. Maura McGrail
(2022)
Cre/lox regulated conditional rescue and inactivation with zebrafish UFlip alleles generated by CRISPR-Cas9 targeted integration
eLife 11:e71478.
https://doi.org/10.7554/eLife.71478

Share this article

https://doi.org/10.7554/eLife.71478

Further reading

    1. Developmental Biology
    Satoshi Yamashita, Shuji Ishihara, François Graner
    Research Article

    Apical constriction is a basic mechanism for epithelial morphogenesis, making columnar cells into wedge shape and bending a flat cell sheet. It has long been thought that an apically localized myosin generates a contractile force and drives the cell deformation. However, when we tested the increased apical surface contractility in a cellular Potts model simulation, the constriction increased pressure inside the cell and pushed its lateral surface outward, making the cells adopt a drop shape instead of the expected wedge shape. To keep the lateral surface straight, we considered an alternative model in which the cell shape was determined by cell membrane elasticity and endocytosis, and the increased pressure is balanced among the cells. The cellular Potts model simulation succeeded in reproducing the apical constriction, and it also suggested that a too strong apical surface tension might prevent the tissue invagination.

    1. Cancer Biology
    2. Developmental Biology
    Sara Jaber, Eliana Eldawra ... Franck Toledo
    Research Article

    Missense ‘hotspot’ mutations localized in six p53 codons account for 20% of TP53 mutations in human cancers. Hotspot p53 mutants have lost the tumor suppressive functions of the wildtype protein, but whether and how they may gain additional functions promoting tumorigenesis remain controversial. Here, we generated Trp53Y217C, a mouse model of the human hotspot mutant TP53Y220C. DNA damage responses were lost in Trp53Y217C/Y217C (Trp53YC/YC) cells, and Trp53YC/YC fibroblasts exhibited increased chromosome instability compared to Trp53-/- cells. Furthermore, Trp53YC/YC male mice died earlier than Trp53-/- males, with more aggressive thymic lymphomas. This correlated with an increased expression of inflammation-related genes in Trp53YC/YC thymic cells compared to Trp53-/- cells. Surprisingly, we recovered only one Trp53YC/YC female for 22 Trp53YC/YC males at weaning, a skewed distribution explained by a high frequency of Trp53YC/YC female embryos with exencephaly and the death of most Trp53YC/YC female neonates. Strikingly, however, when we treated pregnant females with the anti-inflammatory drug supformin (LCC-12), we observed a fivefold increase in the proportion of viable Trp53YC/YC weaned females in their progeny. Together, these data suggest that the p53Y217C mutation not only abrogates wildtype p53 functions but also promotes inflammation, with oncogenic effects in males and teratogenic effects in females.