CRISPR/Cas9-mediated pou4f3 knockout induces defects in the development of the zebrafish inner ear

Abstract Objective: The zebrafish is an excellent model for studying gene function in auditory system development. Pou4f3 plays an important role in mouse hair cell formation. Here, we constructed a pou4f3-knockout Tg(Brn3c:GFP) zebrafish to provide an efficient fluorescence-visualized model for studying the molecular mechanisms of ear development. Methods: Cas9/single guide RNAs targeting exon 2 of pou4f3 were designed and injected into one-cell stage zebrafish embryos (G0 generation). The G0 generation were crossed with Tg(Brn3c:GFP) zebrafish to obtain pou4f3-mutant Tg(Brn3c:GFP) zebrafish. The targeting efficiency was detected by polymerase chain reaction amplification and Sanger sequencing. Zebrafish hair cells were observed by laser scanning confocal microscopy in vivo. The morphology of the otoliths and semicircular canals were analyzed. All animal experiments were approved by the Animal Care and Use Committee of Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University (approval No. 2016-KY-040) on March 3, 2016. Results: The pou4f3-mutant Tg(Brn3c:GFP) zebrafish line was successfully established. Fluorescence observation suggested that hair cell development was delayed in pou4f3-knockout zebrafish. Knockout of pou4f3 also induced defects in the otoliths and semicircular canals and impaired ear function in zebrafish. Conclusion: A CRISPR/Cas9-mediated pou4f3 mutant Tg(Brn3c:GFP) zebrafish model was established for the first time to demonstrate the essential role of pou4f3 in zebrafish ear development. Our study provides a highly efficient method for the establishment of a visualized model of gene knockout zebrafish and has the potential to allow high-throughput drug screening to explore therapeutics for related diseases.