# Precision base-editing of the cryptic 3' acceptor site to correct the RNA splicing defect of β654-thalassemia Article · Lu D, Gong X, Guo X, Cai Q, Li W, Chen Y, Li D, Zeng F - Archive id: doi:10.1186/s43556-026-00589-z - Published: 2026-09-30 - Venue: Molecular biomedicine - DOI: https://doi.org/10.1186/s43556-026-00589-z - PMID: 42814316 - PMCID: PMC13627643 - Citations: 0 - Source URL: https://doi.org/10.1186/s43556-026-00589-z - PDF: https://europepmc.org/articles/PMC13627643/pdf - Provenance: europepmc ## Abstract β-Thalassemia is one of the most common inherited disorders worldwide and is caused by mutations affecting β-globin production. β 654 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 β 654 -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 β 654 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 β 654 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 β 654 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 β 654 -thalassemia. _Abstract reproduced under CC-BY (article license as recorded by Europe PMC; abstract reproduced unmodified apart from markup removal)._ Topics: [CRISPR & Gene-Editing Therapies](https://space-kitty.com/topics/gene-editing-therapies) --- Space Kitty Archive · https://space-kitty.com · API: https://space-kitty.com/api/v1 · MCP: https://space-kitty.com/mcp · Premium: $0.20/request via x402 (Base Sepolia (testnet)). Metadata: arXiv (CC0), OpenAlex (CC0), Europe PMC (CC BY/CC0 works), ClinicalTrials.gov.