Ocular phenotypes in CRISPR rlbp1b^-/- zebrafish provide a screening platform for evaluating the pathogenicity of human RLBP1 variants using humanised transgenic lines

Abstract

Purpose : The visual cycle regenerates the visual chromophore 11-cis-retinaldehyde. Cellular retinaldehyde binding protein (CRALBP), encoded by the RLBP1 gene in humans, facilitates this retinoid cycle in retinal pigment epithelium (RPE) and Müller glia cells. RLBP1 is associated with inherited retinal diseases including Newfoundland Rod-Cone Dystrophy and Bothnia dystrophy. Using zebrafish rlbp1b knockouts, we are assessing the role of RPE-expressed Rlbp1b in vision and developing a platform to assess the pathogenicity of patient RLBP1 variants.

Methods : rlbp1b^-/- knockouts were generated using CRISPR-Cas9 editing and humanised lines by Tol2 transgenesis. A human RLBP1 cDNA plasmid from the DNASU repository was used to create Tol2 expression vectors using multisite Gateway cloning. RLBP1 patient variant expression vectors were created using site directed mutagenesis. Transgenic zebrafish lines were created by microinjecting the expression vectors and Tol2 transposase mRNA into eggs from rlbp1b^-/- knockouts. Tol2 excision assays confirmed plasmid integration into the genome. Retinal histology was examined by light microscopy and visual behaviour by the optokinetic response (OKR).

Results : Optokinetic response assays using rlbp1b^-/- larvae indicate a 50% reduction in the number of saccades per min compared to wildtype. Tol2 expression vectors were engineered to express N-terminal GFP-tagged, human wildtype RLBP1 or patient R151Q and A237V variants, under control of an rpe65a promoter. Embryo microinjection yielded 50-60% larvae with fluorescent pineals suggesting successful regulated expression of the human gene in the zebrafish RPE and pineal. F0 GFP-positive larvae were raised to adulthood to screen for germline transmitters.

Conclusions : Here, we show a visual behaviour defect in rlbp1b^-/- zebrafish larvae and describe a system where knockout and transgenic zebrafish technology is used in tandem to create humanised zebrafish lines. Stable transgenic lines will then be used screen the pathogenicity of RLBP1 variants by visual behaviour assays, retinoid biochemistry and retinal histology.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.