Novel CNG Channelopathy Model Generated using CRISPR/Cas9-Mediated Genome Editing in Zebrafish

Abstract

Inherited defects in CNGA3 and CNGB3 genes encoding cone cyclic nucleotide-gated (CNG) channel subunits have been linked to complete and incomplete achromatopsia, progressive cone dystrophy and macular degeneration in humans. Several disease-associated frameshift/truncation mutations are located within the last exon of human CNGA3. To mimic these mutations, we used CRISPR/Cas9-mediated genome editing to target the two zebrafish orthologs of human CNGA3. We edited zebrafish cnga3a and cnga3b using microinjection into single-cell embryos of Cas9 mRNA plus single guide RNAs targeting sites within the last exon of the genes (exon 9 and exon 5, respectively). Successful editing was confirmed via mismatch cleavage using T7 endonuclease I, and DNA sequencing, demonstrating insertions/deletions (indels) at the edited sites. The functional effects of editing were determined using optomotor response (OMR) visual performance assays and electroretinogram (ERG) recordings with 6-8 dpf larvae. Retinal morphology was assessed by immunohistochemistry, while retinal cell death was investigated via TUNEL staining. Indels in cnga3a were associated with a profound disruption of visual function in larvae, demonstrated by impaired OMR performance and attenuated ERG b-wave amplitudes, at a developmental stage when only cone-based vision is present. Compared to editing of cnga3a, CRISPR modification at homologous target sites in cnga3b produced a more modest effect on visual function in larvae, while editing of both cnga3a and cnga3b together completely disrupted visual performance. In addition, adult cnga3a-edited zebrafish exhibited disturbed retinal morphology with disorganized cone photoreceptors and shortened cone outer segments, evidence of retinal cell death, and mislocalized cone CNG channels. Together, these results suggest that cnga3a exon 9 editing in zebrafish represents a novel channelopathy model for human cone dysfunction and degeneration, which recapitulates truncation mutations in human CNGA3 that are associated with blindness.