PUBLICATION
Rapid and robust generation of cardiomyocyte-specific crispants in zebrafish using the cardiodeleter system
- Authors
- Keeley, S., Fernández-Lajarín, M., Bergemann, D., John, N., Parrott, L., Andrea, B.E., González-Rosa, J.M.
- ID
- ZDB-PUB-250327-4
- Date
- 2025
- Source
- Cell reports methods 5: 101003101003 (Journal)
- Registered Authors
- Bergemann, David, Gonzalez-Rosa, Juan Manuel
- Keywords
- CP: developmental biology, CP: genetics, CRISPR-Cas9, crispants, myocardium, tissue-specific mutants, transgenesis
- MeSH Terms
-
- Gene Editing/methods
- Gene Targeting/methods
- CRISPR-Cas Systems*/genetics
- Animals, Genetically Modified
- Animals
- Mutation
- Myocytes, Cardiac*/metabolism
- Zebrafish Proteins/genetics
- Zebrafish Proteins/metabolism
- RNA, Guide, CRISPR-Cas Systems*/genetics
- Zebrafish*/genetics
- PubMed
- 40132543 Full text @ Cell Rep Methods
Citation
Keeley, S., Fernández-Lajarín, M., Bergemann, D., John, N., Parrott, L., Andrea, B.E., González-Rosa, J.M. (2025) Rapid and robust generation of cardiomyocyte-specific crispants in zebrafish using the cardiodeleter system. Cell reports methods. 5:101003101003.
Abstract
CRISPR-Cas9 has accelerated loss-of-function studies in zebrafish, but creating tissue-specific mutant lines is still labor intensive. While some tissue-specific Cas9 zebrafish lines exist, standardized methods for gene targeting, including guide RNA (gRNA) delivery, are lacking, limiting broader use in the community. To tackle these limitations, we develop a cardiomyocyte-specific Cas9 line, the cardiodeleter, that efficiently generates biallelic mutations in combination with gene-specific gRNAs. We create transposon-based guide shuttles that deliver gRNAs targeting a gene of interest while permanently labeling cells susceptible to becoming mutant. We validate this modular approach by deleting five genes (ect2, tnnt2a, cmlc2, amhc, and erbb2), resulting in the loss of the corresponding protein or phenocopy of established mutants. We provide detailed protocols for generating guide shuttles, facilitating the adoption of these techniques in the zebrafish community. Our approach enables rapid generation of tissue-specific crispants and analysis of mosaic phenotypes, making it a valuable tool for cell-autonomous studies and genetic screening.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping