Bi-allelic pathogenic variants in ABCA4 cause Stargardt disease (STGD1), an autosomal recessive retinal disorder leading to progressive central vision loss, typically beginning in childhood. The disease results from the accumulation of cytotoxic products in photoreceptors due to dysfunctional ABCA4 protein. Among the diverse mutations in ABCA4, the exonic c.768G>T variant is significant and frequent, causing mis-splicing that retains a 35-nucleotide elongation of exon 6 in mature mRNA. This mis-splicing arises from a weakened canonical donor splice site and a strong cryptic splice donor site downstream, leading to a frameshift and a truncated protein.

CRISPR/Cas-mediated genome editing has shown promise in correcting splicing defects. However, no strategies have been established for variants near exon/intron boundaries. To address this, we employed our EDSpliCE (Enhanced-Deletion Splicing Correction Editing) platform to correct the c.768G>T splicing defect. We cloned a mutant minigene plasmid with the ABCA4:c.768G>T variant and confirmed pathogenic splicing through a minigene assay in HEK293T cells. The validated minigene was co-transfected with individual sgRNAs and Cas9-ortholog EDSpliCE. Correctly spliced transcript percentages were analyzed via PCR and chip electrophoresis, with editing efficiency assessed by targeted high-throughput sequencing. Then, to explore top sgRNAs for correcting splicing defects in photoreceptor precursor cells, an isogenic ABCA4:c.768G>T iPSC line was created using CRISPR/Cas9 RNP and a donor template.

Our findings indicate that EDSpliCE can effectively correct splicing defects at exon/intron boundaries, achieving up to 85% correction of the c.768G>T defect, outperforming traditional Cas9 methods. This positions EDSpliCE as a promising therapeutic approach for addressing splicing variants like ABCA4c.768G>T in STGD1.