Abstract: Hearing loss is a widespread global health issue that affects all regions and countries. It is classified into conductive hearing loss, sensorineural hearing loss, and mixed hearing loss. Oxidative stress and redox homeostasis imbalance are important factors causing cochlear damage, and NRF2 is a crucial antioxidant regulatory factor. In the human body, the Nfe2l2 gene is widely expressed in the central nervous system, auditory organs, and metabolism-related organs and tissues, playing a significant role in maintaining cellular redox homeostasis, responding to oxidative stress, and regulating metabolism, inflammation, autophagy, and immune responses. In noise exposure experiments, Nrf2-KO mice showed more severe hearing impairment compared to wild-type mice. In zebrafish, nrf2a is widely expressed in the early stages of embryonic development and acts as a core transcription factor to regulate the expression of a series of antioxidant genes, constituting an important defense mechanism against exogenous oxidative damage from chemicals and heavy metals. Besides its antioxidant function, this gene is crucial for the development and homeostasis maintenance of various organs in zebrafish, such as the liver and nervous system, and plays an important protective role in regulating inflammatory responses, metabolic processes, and inhibiting apoptosis. To study the specific mechanism of this gene in the development of inner ear and lateral line hair cells, a zebrafish nrf2a gene knockout line was constructed using CRISPR/Cas9 gene editing technology. First, online analysis tools were used to screen the knockout target sites of the nrf2a gene, and the guide DNA of this gene was amplified and synthesized by polymerase chain reaction. This was then used as a template for transcription, and the transcribed sgRNA was mixed with Cas9 protein at a ratio of 5: 1 and injected into 1-cell stage zebrafish embryos. Subsequently, the effectiveness of the gene knockout was verified. The results showed that there was a large fragment deletion in the nrf2a gene, and translation was prematurely terminated, indicating successful knockout of the gene. After screening, a homozygous mutant of the nrf2a gene was obtained, laying the foundation for studying the role of this gene in the development of inner ear and lateral line hair cells.
Key words: zebrafish; CRISPR/Cas9; nrf2a gene; hearing loss; inner ear development
（Acta Laser Biology Sinica, 2026, 35(1): 072-080）