Abstract
Missplicing of transcripts is a frequent molecular mechanism in a wide range of inherited genetic conditions. Therapeutic splicing correction can be achieved through antisense oligonucleotides; however, they do not enable permanent correction. Concurrently, CRISPR-Cas9 approaches often rely on dual-guide RNA-induced larger deletions-for instance, pseudoexons removal-which raises concerns about higher genotoxicity from multiple double-strand breaks. We therefore investigated single-guide RNA CRISPR-Cas9 approaches to address the recurrent pathogenic USH2A:c.7595-2144A>G deep-intronic variant. Using single-guide RNAs with either Cas9 or Cas9 fused to TREX2 (EDCas9), we restored correct splicing in a minigene assay and patient-derived fibroblasts. Cas9 with single-guide RNAs generated small indels, but their frequency and extent varied between models, resulting in variable productivity with respect to splicing rescue efficacy. In contrast, EDCas9 produced larger, directional deletions with a consistent profile across both models, effectively disrupting missplicing-inducing sequences and ensuring robust splicing correction. Off-target assessments revealed a safe profile for both Cas9 and EDCas9, with EDCas9 additionally preventing targeted translocations. Virus-like particles delivered EDCas9 and a lead gRNA, demonstrating suitability as a transient delivery system. In conclusion, EDCas9 emerges as a flexible and powerful editing approach for addressing the pathogenic USH2A:c.7595-2144A>G variant, paving the way for further therapeutic investigation.
Keywords:
CRISPR; Cas9; MT: RNA/DNA Editing; TREX2; USH2A; Usher syndrome; VLP; gene editing; indels; splicing.