Gene Editing of the Endogenous Cryptic 3¢ Splice Site Corrects the RNA Splicing Defect in the b⁶⁵⁴-Thalassemia Mouse Model

b⁶⁵⁴-thalassemia is caused by a point mutation in the second intron (IVS-II) of the b-globin gene that activates a cryptic 3¢ splice site, leading to incorrect RNA splicing. Our previous study demonstrated that when direct deletion of the b⁶⁵⁴ mutation sequence or the cryptic 3¢ splice site in the IVS-II occurs, correct splicing of b-globin mRNA can be restored. Herein, we conducted an in-depth analysis to explore a more precise gene-editing method for treating b⁶⁵⁴-thalassemia. A single-base substitution of the cryptic 3¢ acceptor splice site was introduced in the genome of a b⁶⁵⁴-thalassemia mouse model using clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9(Cas9)-mediated homology-directed repair (HDR). All of the HDR-edited mice allow the detection of correctly spliced b-globin mRNA. Pathological changes were improved compared with the nonedited b⁶⁵⁴ mice. This resulted in a more than twofold increase in the survival rate beyond the weaning age of the mice carrying the b⁶⁵⁴ allele. The therapeutic effects of this gene-editing strategy showed that the typical b-thalassemia phenotype can be improved in a dose-dependent manner when the frequency of HDR is over 20%. Our research provides a unique and effective method for correcting the splicing defect by gene editing the reactive splicing acceptor site in a b⁶⁵⁴ mouse model.