Transcriptomics and phenotypic analysis of OTOF gene knockdown in zebrafish mediated by CRISPR/Cas9

Abstract

Deafness is a common genetic disorder, where mutations,in the OTOF gene can disrupt the normal functionof the Otoferlin protein, leading to impaired neurotransmitter release in the inner ear and subsequent deafness. Despite the complexity of the pathogenic mechanism,it is not fully understood. Zebrafish are an excellent model for studying genetically-induced deafness,but there have been no previous reports on the pathogenesis of OTOF in zebrafish.This study successfully established a zebrafish model with mutated OTOF genes using CRISPR/Cas9 gene editing technology to investigate the molecular basis of OTOF-induced deafness. Compared to AB wild type zebrafish, those with low otof expression showed injury and apoptosis of hair cells in the posterior lateral neuromasts along with significant increase in the number of macrophages and apoptotic cells in this region. Additionally, these mutants exhibited a reduction in body length. To further elucidate differences at 5dpf (days post-fertilization) between mutant and wild type zebrafish embryos, RNA-seq analysis was conducted to examine differentially expressed genes (DEGs).A total of 334 up-regulated DEGs and 111 down-regulated DEGs were identified in mutants compared to wild types.KEGG and GO enrichment analyses were performed on these DEGs to identify key signaling pathways and hub DEGs. The findings revealedan increased expression of several genes involved in the HSP70 oxidative stress system, suggesting that OTOF may protect cochlear hair cell from apoptosis induced by oxidative stress through regulation of MAPK signal and HSP70 expression.In summary, the establishment of a zebrafish model with OTOF knockout provides a valuable tool for investigating the function of Otoferlin and understanding the role of the OTOF gene in deafness. These potential molecular insights offer significant contributions towards understanding the pathogenesis of deafness experimental models and serves as a foundation for comprehending the involvement of the OTOF gene.