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.

Reviewing Editor

  1. Tanya T Whitfield, University of Sheffield, United Kingdom

Publication history

  1. Preprint posted: June 18, 2021 (view preprint)
  2. Received: June 20, 2021
  3. Accepted: June 16, 2022
  4. Accepted Manuscript published: June 17, 2022 (version 1)
  5. Version of Record published: July 8, 2022 (version 2)

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.

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  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

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