Fig 1

Fig 1 Development of USH3A zebrafish model.

CRISPR/Cas9 technology was used to generate a large deletion within the clrn1 gene and establish a germ-line transmitting mutant line. Pairs of CRISPR gRNAs were designed to target Exon 1 and the 3’ UTR of clrn1. (a) Depiction of CRISPR cut sites in relation to clrn1 zebrafish exons and protein functional domains. (b) Method to mosaically mutate the tyrosinase gene and deplete pigmentation from the retinal pigment epithelium to allow for RNAscope probe detection. (c) Detection of clrn1 transcripts (bright red dots) in the inner retina of 1 mpf wild-type zebrafish using the highly sensitive RNAscope in-situ hybridization assay. Retinal Pigment Epithelium (RPE), Outer Nuclear Layer (ONL), Inner Nuclear Layer (INL), and Ganglion Cell Layer (GCL) are labeled in (b) and (c). (d) Phalloidin (actin, green) and Hoechst (nuclei, magenta) staining on 7dfp wild-type and clrn1^-/- larvae to assess inner ear hair cell structure. Red arrow indicates pycnotic nuclei. n=15/15 embryos showing normal stereocilia in wild-type and 15/15 embryos showing splayed sterocilia in clrn1-/- mutants. Schematic in (b) created in BioRender under License: https://BioRender.com/c00b794.