(Laboratory of Animal Nutrition and Human Health, Hunan Normal University, Changsha 410081, China)

Abstract: β-tectorin, encoded by the TECTB gene, plays an important regulatory role in human hearing function, is an important component of the covering membrane in the inner ear organ, and plays a key role in the process of sound transduction into neural signals. The mutation of β-tectorin can lead to the occurrence of non-syndromic deafness in humans. However, the molecular mechanism of hearing impairment remains unclear. To study the role of the tectb gene in zebrafish inner ear development, this study successfully constructed a zebrafish model with tectb gene deletion using CRISPR/Cas9 gene editing technology and conducted a preliminary analysis of the phenotype. First, target sites and genotype identification primers were designed on exon 3 of tectb gene. Guide DNA (sgDNA) was amplified in vitro, next guide RNA (sgRNA) was obtained by in vitro transcription. Then, sgRNA and Cas9 enzymes were co-injected into wild-type zebrafish embryos by microinjection. After genotyping, the mutant chimera F0 generation fish were screened; and after being raised to adult, they were crossed with wild-type zebrafish to obtain the mutant heterozygotes of the F1. After genetic stability identification and sequencing, F1-generation fish with frameshift mutation were raised to adulthood, they were crossed to obtain F2 tectb homozygotes. Finally, through observation and hair cell staining experiments, it was found that the inner ear structure of the homozygotes mutant zebrafish appears normal, with no obvious difference in the shape and size of the otolith, and no obvious defect in the development of hair cells. However, whether there were changes in the development of internal organs and tissues should be further studied. This study provides us with a research model to gain insight into the molecular mechanisms of inner ear development and human diseases. With the help of the tectb gene knockout zebrafish model, we can elucidate the role of this gene in diseases and better understand the pathways involved in inner ear development, which is conducive to further research on the regulatory mechanism of hearing.

Key words: zebrafish; tectb; CRISPR/Cas9; inner ear development; hair cells

（Acta Laser Biology Sinica, 2024, 33(2): 143-150）