EDSpliCE, a CRISPR-Cas9 gene editing platform to rescue splicing, effectively corrects inherited retinal dystrophy-associated splicing defects

Background: Correct splicing of transcripts is essential to ensure the production of functional gene products in eukaryotic cells. Missplicing of transcripts has been identified as the underlying molecular mechanisms behind various disease-causing variants in a wide range of inherited genetic conditions. Achieving therapeutic splicing correction is possible through antisense oligonucleotide and CRISPR/Cas9 strategies. However, while antisense oligonucleotides offer effective modulation, they do not enable for permanent correction. On the other hand, current CRISPR/Cas9 approaches often rely on dual-gRNA-inducing deletion of larger pieces of DNA, containing the site(s) responsible for the splicing defect, particularly the elimination of pseudoexons, raising concerns about potential chromosomal instability. Results: The novel gene editing strategy, Enhanced-Deletion Splicing Correction Editing (EDSpliCE), just uses single gRNAs to effectively correct aberrant splicing caused by pseudoexon sequence inclusion into the mature mRNA. By employing Cas9 fused to a human exonuclease (TREX2), EDSpliCE achieves targeted enhanced deletions of sequences involved in pseudoexon recognition, thereby restoring correct splicing of the pre-mRNA. By addressing two isolated (ABCA4:c.5197-557G>T and USH2A:c.7595-2144A>G) and two clustered (ABCA4:c.5196+1013A>G and ABCA4:c.5196+1056A>G) pathogenic deep-intronic variants, we demonstrated effective splicing rescue in minigene assay employing distinct single gRNAs. Further validation in patient-derived fibroblasts for the common USH2A:c.7595-2144A>G variant confirmed consistent and high splicing correction. Additionally, the characterization of achieved gene editing affirmed the generation of enhanced deletions by EDSpliCE, revealed high directionality of editing events for all the single gRNAs tested in patient-derived fibroblasts and did not show higher off-target editing potential on selected loci. Conclusions: The successful implementation of the EDSpliCE platform for splicing correction and modulation offers a promising and versatile gene editing approach to address splicing defects, potentially providing a safer option to existing gene editing strategies.