Abstract
Purpose :
Mutations in the retinoid flippase ABCA4 are associated with multiple blinding disorders, including Stargardt disease, retinitis pigmentosa, and age-related macular degeneration (AMD). ABCA4 removes toxic all-trans retinal from photoreceptor discs to prevent its conversion to toxic lipofuscin. Researchers often use Abca4 mutant mice to study the effects of associated eye disease; however, this line does not recapitulate the human disease phenotype unless combined with Rdh8 mutants. Furthermore, mice are a cone-poor species, and their retinas do not reflect the cone-exclusive environment of the human fovea. Inactivation of ABCA4 orthologs in a cone-rich diurnal animal model will provide valuable insight into its role in Stargardt disease and other retinopathies in the visually cone-dominant human retina.
Methods :
Zebrafish abca4a and abca4b genes were inactivated using CRISPR/Cas9 methods. Guide RNAs were designed using CCTop, and synthesized by Synthego.
Results :
Six individual mutant alleles were generated for zebrafish abca4a, and two for abca4b. Three abca4a alleles were predicted to disrupt RNA splicing, two cause inframe deletion of short amino acid sequences, and one led to premature stop codons. Both abca4b alleles caused premature stop codons.
Conclusions :
The abca4a and abca4b mutant alleles will be a powerful platform to study loss of ABCA4 function in the cone-rich zebrafish. Development of a monogenic ABCA4 disease model will advance understanding of Stargardt disease, AMD and other retinopathies, and will facilitate development of therapeutic interventions to prevent vision loss. Future work includes generation of abca4a abca4b double homozygous zebrafish to characterize the retinal phenotype, as well as assessment of lipofuscin accumulation and photoreceptor health.
This is a 2020 ARVO Annual Meeting abstract.