Abstract: microRNAs (miRNAs), a type of small non-coding RNA widely involved in various physiological processes, regulates the expression of protein-coding genes by binding to target mRNA. The miR-194 gene is highly expressed in the liver and serves as a key serum biomarker for diagnosing early liver injury in humans. However, the role of miR-194 in the progression of liver injury remains unclear. The results of bioinformatics analysis indicated that the miR-194 gene was highly conserved in various species. To reveal the role of miR-194 gene in liver development, this study utilized CRISPR/Cas9 gene editing technology to construct miR-194b knockout lines, and to preliminarily investigate the role of miR-194b gene in liver development. First, two target sites of miR-194b gene were designed using an online website. The sgRNA template DNA was obtained by PCR amplification, and then DNA was transcribed to generate sgRNA. The sgRNA and Cas9 protein were co-injected into the zebrafish 1-cell embryos. After injection, the embryos were raised to 2 months of age and screened to obtain the F0 generation chimeric mutants. F0 chimeras were crossed with wild-type zebrafish to obtain F1 heterozygotes, which were subsequently genotyped and screened for the same mutation type by Sanger sequencing. F1 generation heterozygotes were crossed and genotyped to obtain F2 generation of homozygous mutants. The miR-194b homozygous mutants exhibited no obvious phenotypic abnormalities and were fertile. Overexpression of miR-194b caused does-dependent developmental malformations, including disorganized myotomes, pericardial edema, smaller liver, and absence of swim bladder, demonstrating that the miR-194b plays an important role in the early development and organogenesis of zebrafish embryos. In this study, we successfully constructed a zebrafish miR-194b knockout line, which lays the foundation for an in-depth study of the role of miR-194 in liver development.
Key words: zebrafish; miR-194b gene; liver development; microRNAs; CRISPR/Cas9
（Acta Laser Biology Sinica, 2025, 34(4): 319-327）