Generation of Knockout Zebrafish using CRISPR/Cas9 for the Study of Development and Disease Mechanisms.

INTS14/VWA9, a component of the integrator complex subunits, plays a pivotal role in regulating the fate of numerous nascent RNAs transcribed by RNA polymerase II, particularly in the biogenesis of small nuclear RNAs and enhancer RNAs. Despite its significance, a comprehensive mutation model for developmental research has been lacking. To address this gap, we aimed to investigate the expression patterns of during zebrafish embryonic development. We generated mutant strains using the CRISPR/Cas9 system. We validated the gRNA activity by co-injecting Cas9 protein and a single guide RNA into fertilized zebrafish eggs, subsequently confirming the presence of a 6- or 9-bp deletion in the gene. In addition, we examined the two mutant alleles through PCR analysis, T7E1 assay, TA-cloning, and sequencing. For the first time, we used the CRISPR/Cas9 system to create a model in which some sequences of the gene were removed. This breakthrough opens new avenues for in-depth exploration of the role of in animal diseases. The mutant strains generated in this study can provide a valuable resource for further investigations into the specific consequences of gene deletion during zebrafish development. This research establishes a foundation for future studies exploring the molecular mechanisms underlying the functions of , its interactions with other genes or proteins, and its broader implications for biological processes.