Precision base-editing of the cryptic 3' acceptor site to correct the RNA splicing defect of β⁶⁵⁴-thalassemia

β-Thalassemia is one of the most common inherited disorders worldwide and is caused by mutations affecting β-globin production. β⁶⁵⁴ mutation (IVS2-654, C > T) is one of the most frequently occurring β-thalassemia alleles in Han Chinese population, which activates a cryptic 3' splice site and leads to aberrant RNA splicing. Previous studies demonstrated that direct base editing of the IVS2-654 mutation is challenging because suitable single-guide RNAs (sgRNAs) cannot effectively target this site. Here we investigated an alternative therapeutic strategy by targeting the mutation-activated cryptic 3' splice acceptor site, rather than the disease-causing mutation itself. We first introduced base substitutions into the cryptic splice acceptor site in a β⁶⁵⁴-thalassemia mouse model using CRISPR-Cas9-mediated homology-directed repair, which restored normal RNA splicing and validated the therapeutic rationale of this approach. We then generated base-edited β⁶⁵⁴ mice by microinjecting Td-CBEmax mRNA together with sgRNAs targeting the cryptic splice acceptor site into one-cell embryos. Base editing was successfully achieved in 78% of live-born β⁶⁵⁴ mice, of which 86% produced correctly spliced β-globin transcripts. Restoration of normal RNA splicing was accompanied by marked improvement of hematological parameters and tissue pathology in most base-edited founder mice and their offspring compared with non-edited β⁶⁵⁴ mice. Together, these findings demonstrate that mutation-activated cryptic splice sites are therapeutically actionable targets for precision base editing and provide proof of concept for a precise and effective strategy to correct aberrant RNA splicing in β⁶⁵⁴-thalassemia.