Correction of RNA splicing defect in β(654)-thalassemia mice using CRISPR/Cas9 gene-editing technology

β(654)-thalassemia is a prominent Chinese subtype of β-thalassemia, representing 17% of all cases of β-thalassemia in China. The molecular mechanism underlying this subtype involves the IVS-2-654 C→T mutation leading to aberrant β-globin RNA splicing. This results in an additional 73-nucleotide exon between exons 2 and 3 and leads to a severe thalassemia syndrome. Herein, we explored a CRISPR/Cas9 genome editing approach to eliminate the additional 73-nucleotide by targeting both the IVS-2-654 C→T and a cryptic acceptor splice site at IVS-2-579 in order to correct aberrant β-globin RNA splicing and ameliorate the clinical β-thalassemia syndrome in β(654) mice. Gene-edited mice were generated by microinjection of sgRNA and Cas9 mRNA into one-cell embryos of β(654) or control mice: 83.3% of live-born mice were gene-edited, 70% of which produced correctly spliced RNA. No off-target events were observed. The clinical symptoms, including hematologic parameters and tissue pathology of all of the edited β(654) founders and their offspring were significantly improved compared to those of the non-edited β(654) mice, consistent with the restoration of wild-type β-globin RNA expression. Notably, the survival rate of gene-edited heterozygous β(654) mice increased significantly, and live-born homozygous β(654) mice were observed. Our study demonstrated a new and effective gene-editing approach that may provide groundwork for the exploration of β(654)-thalassemia therapy in the future.