PUBLICATION
Conditional, Tissue-Specific CRISPR/Cas9 Vector System in Zebrafish Reveals the Role of Nrp1b in Heart Regeneration
- Authors
- Singh Angom, R., Wang, Y., Wang, E., Dutta, S., Mukhopadhyay, D.
- ID
- ZDB-PUB-230831-57
- Date
- 2023
- Source
- Arteriosclerosis, Thrombosis, and Vascular Biology 43(10): 1921-1934 (Journal)
- Registered Authors
- Dutta, Shamit, Mukhopadhyay, Debabrata, Wang, Enfeng, Wang, Ying
- Keywords
- clustered regularly interspaced short palindromic repeats, myocytes, cardiac, neuropilin-1, regeneration, zebrafish
- MeSH Terms
-
- Animals
- CRISPR-Cas Systems*
- Gene Editing
- Myocytes, Cardiac*/physiology
- Neuropilin-1/genetics
- Regeneration*
- Ventricular Remodeling
- Zebrafish*/genetics
- Zebrafish Proteins*/physiology
- PubMed
- 37650323 Full text @ Arterio., Thromb., and Vas. Bio.
Citation
Singh Angom, R., Wang, Y., Wang, E., Dutta, S., Mukhopadhyay, D. (2023) Conditional, Tissue-Specific CRISPR/Cas9 Vector System in Zebrafish Reveals the Role of Nrp1b in Heart Regeneration. Arteriosclerosis, Thrombosis, and Vascular Biology. 43(10):1921-1934.
Abstract
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 nrp1 isoforms, including nrp1a and nrp1b, 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 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.
Results We 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.
Conclusions To our knowledge, this is the first study 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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping