The gut microbiota constitutes a complex ecosystem that has an important impact on host health. In this study, genetically engineered zebrafish with inducible nitric oxide synthase (iNOS or NOS2) knockout were used as a model to investigate the effects of nos2a/nos2b gene single knockout and nos2 gene double knockout on intestinal microbiome composition and function. Extensive 16S rRNA sequencing revealed substantial changes in microbial diversity and specific taxonomic abundances, yet it did not affect the functional structure of the intestinal tissues. Notably, iNOS-deficient zebrafish demonstrated a decrease in Vibrio species and an increase in Aeromonas species, with more pronounced effects observed in double knockouts. Further transcriptomic analysis of the gut in double iNOS knockout zebrafish indicated significant alterations in immune-related and metabolic pathways, including the complement and PPAR signaling pathways. These findings underscore the crucial interplay between host genetics and gut microbiota, indicating that iNOS plays a key role in modulating the gut microbial ecology, host immune system, and metabolic responses.

The rapid evolution of CRISPR/Cas technology has transformed genome editing across biological systems in which zebrafish have emerged as a powerful vertebrate model for functional genomics and disease research. Due to its transparency, genetic similarity to humans, and suitability for large-scale screening, zebrafish is an appropriate system for translating molecular discoveries into biomedical and environmental applications. Thereby, this review highlights the recent progress in zebrafish gene editing, targeting innovations in ribonucleoprotein delivery, PAM-flexible Cas variants, and precision editors. These approaches have greatly improved editing accuracy, reduced mosaicism, and enabled efficient F0 phenotyping. In the near future, automated microinjections, optimized guide RNA design, and multi-omics validation pipelines are expected to enhance reproducibility and scalability. Although recent innovations such as ribonucleoprotein delivery, PAM-flexible Cas variants, and precision editors have expanded the zebrafish genome-editing toolkit, their benefits are often incremental and context-dependent. Mosaicism, allele complexity, and variable germline transmission remain common, particularly in F0 embryos. Precision editors enable defined nucleotide changes but typically exhibit modest efficiencies and locus-specific constraints in zebrafish. Consequently, rigorous validation, standardized workflows, and careful interpretation of F0 phenotypes remain essential. This review critically examines both the capabilities and limitations of current zebrafish gene-editing technologies, emphasizing experimental trade-offs, reproducibility challenges, and realistic use cases.

The development of intermuscular bones (IBs) in teleost fish is regulated by osteogenic genes, but the upstream signals that initiate ossification remain elusive. Given the spatiotemporal correlation between IB formation and swimming behavior, mechanical stimuli from muscle activity have been hypothesized as potential regulators. In this study, we investigated the role of the muscle-specific microRNA miR499 in this process. Using CRISPR/Cas9-generated miR499 knockout zebrafish, we demonstrated that the mutant of miR499 induces a fast-twitch muscle phenotype through the upregulation of sox6, accompanied by increased fast-myosin gene expression and fast-twitch fiber proliferation. This shift led to distinct locomotor alterations, characterized by reduced tail-beat frequency and increased stride length. Crucially, these changes resulted in delayed IB ossification onset, slower ossification rates, and simplified morphology. The total ossification area decreased by 27%. Molecular analysis revealed a downregulation of osteogenic genes in IB tissue. Our findings establish miR499 as a key regulator of a muscle–bone functional axis, where miRNA-mediated muscle fiber specification determines mechanical output that subsequently guides IB development. This work provides new insights into the mechanobiological regulation of skeletal development and identifies a promising genetic target for aquaculture breeding programs aimed at attenuated IBs.