Gene Editing of the Endogenous Cryptic 3' Splice Site Corrects the RNA Splicing Defect in the β 654 -Thalassemia Mouse Model.

β ⁶⁵⁴ -thalassemia is caused by a point mutation in the second intron (IVS-II) of the β-globin gene that activates a cryptic 3' splice site, leading to incorrect RNA splicing. Our previous study demonstrated that when direct deletion of the β ⁶⁵⁴ mutation sequence or the cryptic 3' splice site in the IVS-II occurs, correct splicing of β-globin mRNA can be restored. Herein, we conducted an in-depth analysis to explore a more precise gene-editing method for treating β ⁶⁵⁴ -thalassemia. A single-base substitution of the cryptic 3' acceptor splice site was introduced in the genome of a β ⁶⁵⁴ -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 β-globin mRNA. Pathological changes were improved compared with the nonedited β ⁶⁵⁴ mice. This resulted in a more than twofold increase in the survival rate beyond the weaning age of the mice carrying the β ⁶⁵⁴ allele. The therapeutic effects of this gene-editing strategy showed that the typical β-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 β ⁶⁵⁴ mouse model.