Cas9-Based Strategies to Rescue Deep-Intronic Mutations in ABCA4

Stargardt disease (STGD) is an autosomal recessive inherited retinal disorder (IRD) caused by biallelic mutations in the ABCA4 gene. IRDs and STGD caused by mutations in ABCA4 are one of the most common forms with an estimated incidence of 1: 10,000. To date, more than 1000 different mutations have been classified as pathogenic. In a considerable number of STGD cases, deep-intronic mutations have been shown to be causative as they result in mRNA mis-splicing. These mRNA-splicing defects lead to either pseudoexon formation or exon skipping which in turn disrupt the normal sequence of codons by frameshift mutations or premature stop codon insertion. As most sequences encompassing deep-intronic mutations have no essential regulatory function, Cas9-based editing exploiting either non-homologous DNA end joining (NHEJ), or Cas9 base editors themselves, offer a potential strategy to rescue deep-intronic mutations. In this project, we apply, assess and compare several Cas9-based approaches to rescue such splicing defects. Additionally, in order to easily assess the different strategies, we also establish fluorescence-reporter vectors which enable to sense the rescue of the deep-intronic mutations back to correct splicing. Preliminary experiments have shown promising results supporting the efficacy and scalability of the tested Cas9-based strategies which can be potentially implemented to target nearly any deep-intronic mutations across the genome.