Dataset Information

A robust and flexible CRISPR/Cas9-based system for neutrophil-specific gene inactivation in zebrafish.

ABSTRACT: CRISPR/Cas9-based tissue-specific knockout techniques are essential for probing the functions of genes in embryonic development and disease using zebrafish. However, the lack of capacity to perform gene-specific rescue or live imaging in the tissue-specific knockout background has limited the utility of this approach. Here, we report a robust and flexible gateway system for tissue-specific gene inactivation in neutrophils. Using a transgenic fish line with neutrophil-restricted expression of Cas9 and ubiquitous expression of single guide (sg)RNAs targeting rac2, specific disruption of the rac2 gene in neutrophils is achieved. Transient expression of sgRNAs targeting rac2 or cdk2 in the neutrophil-restricted Cas9 line also results in significantly decreased cell motility. Re-expressing sgRNA-resistant rac2 or cdk2 genes restores neutrophil motility in the corresponding knockout background. Moreover, active Rac and force-bearing F-actins localize to both the cell front and the contracting tail during neutrophil interstitial migration in an oscillating fashion that is disrupted when rac2 is knocked out. Together, our work provides a potent tool that can be used to advance the utility of zebrafish in identifying and characterizing gene functions in a tissue-specific manner.

SUBMITTER: Wang Y

PROVIDER: S-EPMC8084575 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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A robust and flexible CRISPR/Cas9-based system for neutrophil-specific gene inactivation in zebrafish.

Wang Yueyang Y Hsu Alan Y AY Walton Eric M EM Park Sung Jun SJ Syahirah Ramizah R Wang Tianqi T Zhou Wenqing W Ding Chang C Lemke Abby Pei AP Zhang GuangJun G Tobin David M DM Deng Qing Q

Journal of cell science 20210422 8

CRISPR/Cas9-based tissue-specific knockout techniques are essential for probing the functions of genes in embryonic development and disease using zebrafish. However, the lack of capacity to perform gene-specific rescue or live imaging in the tissue-specific knockout background has limited the utility of this approach. Here, we report a robust and flexible gateway system for tissue-specific gene inactivation in neutrophils. Using a transgenic fish line with neutrophil-restricted expression of Cas ...[more]

PMID: 33722979

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Project description:BackgroundCRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) technology-mediated genome editing has significantly improved the targeted inactivation of genes in vitro and in vivo in many organisms. Neuropilins play crucial roles in zebrafish heart regeneration, heart failure in mice, and electrical remodeling after myocardial infarction in rats. But the cell-specific functions of nrp1 have not been described before. In this study, we have investigated the role of nrp1 isoforms, including nrp1a and nrp1b, in cardiomyocytes during cardiac injury and regeneration in adult zebrafish hearts.MethodsIn this study, we have reported a novel CRISPR-based vector system for conditional tissue-specific gene ablation in zebrafish. Specifically, the cardiac-specific cmlc2 promoter drives Cas9 expression to silence the nrp1 gene in cardiomyocytes in a heat-shock inducible manner. This vector system establishes a unique tool to regulate the gene knockout in both the developmental and adult stages and hence widens the possibility of loss-of-function studies in zebrafish at different stages of development and adulthood. Using this approach, we investigated the role of neuropilin isoforms nrp1a and nrp1b in response to cardiac injury and regeneration in adult zebrafish hearts.ResultsWe observed that both the isoforms (nrp1a and nrp1b) are upregulated after the cryoinjury. Interestingly, the nrp1b knockout significantly delayed heart regeneration and impaired cardiac function in the adult zebrafish after cryoinjury, demonstrated by reduced heart rate, ejection fractions, and fractional shortening. In addition, we show that the knockdown of nrp1b but not nrp1a induces activation of the cardiac remodeling genes in response to cryoinjury.ConclusionsTo our knowledge, this study is novel where we have reported a heat-shock-mediated conditional knockdown of nrp1a and nrp1b isoforms using CRISPR/Cas9 technology in the cardiomyocyte in zebrafish and furthermore have identified a crucial role for the nrp1b isoform in zebrafish cardiac remodeling and eventually heart function in response to injury.

Dataset Information

A robust and flexible CRISPR/Cas9-based system for neutrophil-specific gene inactivation in zebrafish.

Publications

A robust and flexible CRISPR/Cas9-based system for neutrophil-specific gene inactivation in zebrafish.

Similar Datasets

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