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

Author:

Liu Fang1,Kambakam Sekhar1ORCID,Almeida Maira P12,Ming Zhitao1,Welker Jordan M12,Wierson Wesley A13,Schultz-Rogers Laura E12,Ekker Stephen C4ORCID,Clark Karl J4ORCID,Essner Jeffrey J12ORCID,McGrail Maura12ORCID

Affiliation:

1. Department of Genetics, Development and Cell Biology, Iowa State University

2. Interdepartmental Graduate Program in Genetics and Genomics, Iowa State University

3. Interdepartmental Graduate Program in Molecular, Cellular and Developmental Biology, Iowa State University

4. Department of Biochemistry and Molecular Biology, Mayo Clinic

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.

Funder

NIH Office of the Director

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Publisher

eLife Sciences Publications, Ltd

Subject

General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Medicine,General Neuroscience

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