Conditional, Tissue-Specific CRISPR/Cas9 Vector System in Zebrafish Reveals the Role of Nrp1b in Heart Regeneration.

BACKGROUND

CRISPR/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 isoforms, including and , in cardiomyocytes during cardiac injury and regeneration in adult zebrafish hearts.

METHODS

In this study, we have reported a novel CRISPR-based vector system for conditional tissue-specific gene ablation in zebrafish. Specifically, the cardiac-specific promoter drives Cas9 expression to silence the 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 and in response to cardiac injury and regeneration in adult zebrafish hearts.

RESULTS

We observed that both the isoforms ( and ) are upregulated after the cryoinjury. Interestingly, the 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 but not induces activation of the cardiac remodeling genes in response to cryoinjury.

CONCLUSIONS

To our knowledge, this study is novel where we have reported a heat-shock-mediated conditional knockdown of and isoforms using CRISPR/Cas9 technology in the cardiomyocyte in zebrafish and furthermore have identified a crucial role for the isoform in zebrafish cardiac remodeling and eventually heart function in response to injury.