Abstract
Purpose :
ABCA4-retinopathy (including Stargardt disease, STGD1) is by far the most common single-gene inherited retinal disease (IRD). Entire ABCA4 sequencing in a large cohort of over 1,000 STGD1 cases demonstrated the importance of non-coding, mainly deep intronic splicing variants in ABCA4-associated disease pathogenesis. This raises the hypothesis that variants in other non-coding regions, such as cis-regulatory elements (CREs), may be implicated in missing heritability of ABCA4-associated disease. Previous studies have shown that CRISPR/Cas9 editing can be used to disrupt CREs in model organisms. Xenopus (X.) tropicalis is an interesting model organism for IRD, having the major cell types of the human eye, thousands of eggs that are easy to manipulate for CRISPR/Cas9 injections, and a true diploid genome. Here, we aimed to map and functionally study CREs of the abca4 region in X. tropicalis. Moreover, we aimed to generate and characterize a stable knock-out of a CRE of abca4 in X. tropicalis using CRISPR/Cas9 editing.
Methods :
Putative CREs of abca4 were determined according to epigenetic markers H3K4me1 and Pol II in X. tropicalis whole embryo. Regulatory activity of putative CREs was tested using in vitro luciferase assays. Paired guide RNAs (gRNA) and Cas9 in X. tropicalis embryos was used to create a deletion of a selected CRE.
Results :
A putative CRE of abca4, showing around 2-fold increase in luciferase activity compared to empty vector was selected as a target for disruption. Two gRNAs were designed as flanking the target CRE of abca4. The genomic region flanking the CRISPR target site was amplified and sequenced. Genome editing using a paired gRNA CRISPR/Cas9 system showed the deletion of the target CRE compared to using one gRNA and/or non-injected embryos. Further assessments including histology, immunohistochemistry, TUNEL assays and electroretinography to characterize the disease phenotype are ongoing.
Conclusions :
In conclusion, regulatory elements can be disrupted in model organisms using paired gRNAs. Regulatory animal models may contribute to the annotation of the non-coding genome and provide insights into the regulation of IRD genes such as abca4.
This is a 2021 ARVO Annual Meeting abstract.