Abstract
Purpose :
Mutations in CNGA3 and CNGB3 genes encoding cone CNG channels are linked to inherited cone dystrophy/dysfunction in humans, yet the underlying mechanisms taking place within cones in response to these defects are incompletely understood. Our goal is to model cone dysfunction and degeneration using genetic manipulations of cone-rich zebrafish to investigate molecular features that ultimately might be manipulated for rescue of cones.
Methods :
We have used CRISPR/Cas9 genome editing in zebrafish to generate novel models for human blindness associated with mutations in CNGA3. We employed a simple inducible system for Cas9 expression (hsp70:Cas9), combined with constitutive expression of sgRNAs (U6:sgRNA-4(cnga3a/b)), to disrupt the zebrafish orthologs of CNGA3: cnga3aand cnga3b. This allows for normal development of healthy zebrafish retinas and working cone vision prior to gene disruption. We have characterized the functional consequences of these manipulations in zebrafish larvae via optomotor response (OMR) visual performance assays and electroretinogram (ERG) recordings.
Results :
Prior to heat shock (HS+) induction of Cas9 expression at 6 dpf, Tg(hsp70:Cas9; U6:sgRNA-4(cnga3a/b)) larvae displayed OMR performance indistinguishable from wild-type larvae: OMR(+) 80 +/- 4% (SEM, n = 13), compared to 81 +/- 2%, respectively). In contrast, ubiquitous expression of Cas9 (ubi:Cas9) with identical sgRNAs produced larvae at 6 dpf that were only 24 +/- 3% (n = 7) OMR(+). At 3 days following heat shock, Tg(hsp70:Cas9; U6:sgRNA-4(cnga3a/b)) larvae were 37 +/- 12% (n = 7) OMR(+). Both HS+/WT larvae and HS-/Tg+ (hsp70:Cas9; U6:sgRNA-4(cnga3a/b)) control larvae displayed normal OMR performance at 11 dpf. Disruption of visual function after induced editing was confirmed by ERG recordings, which revealed attenuated responses. Consistent with ongoing turnover of the channels, both western blotting and proximity ligation assays (PLA) suggested ubiquitination of native cone photoreceptor CNG channels.
Conclusions :
Dramatic loss of vision 3-4 days following induced disruption of cnga3a/b implies that turnover of functional CNG channels may occur more rapidly than that expected via canonical RPE phagocytosis of outer segment biomolecules. Furthermore, our results indicate that the dramatic visual phenotype of cnga3a/b-edited zebrafish does not depend alone on disruption of retinal development.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.