Optimization of methods for rapid and robust generation of cardiomyocyte-specific crispants in zebrafish using the...

CRISPR/Cas9 has massively accelerated the generation of gene loss-of-function models in zebrafish. However, establishing tissue-specific mutant lines remains a laborious and time-consuming process. Although a few dozen tissue-specific Cas9 zebrafish lines have been developed, the lack of standardization of some key methods, including gRNA delivery, has limited the implementation of these approaches in the zebrafish community. To tackle these limitations, we have established a cardiomyocyte-specific Cas9 line, the cardiodeleter , which efficiently generates biallelic mutations in combination with gene-specific gRNAs. We have also optimized the development of transposon-based guide shuttles that carry gRNAs targeting a gene of interest and permanently label the cells susceptible to becoming mutant. We validated this modular approach by deleting five genes ( ect2 , tnnt2a , cmlc2 , amhc , and erbb2 ), all resulting in the loss of the corresponding protein or phenocopying established mutants. Additionally, we provide detailed protocols describing how to generate guide shuttles , which will facilitate the dissemination of these techniques in the zebrafish community. Our approach enables the rapid generation of tissue-specific crispants and analysis of mosaic phenotypes, bypassing limitations such as embryonic lethality, making it a valuable tool for cell-autonomous studies and genetic screenings.