Correction of RNA splicing defect in β654-thalassemia mice using CRISPR/Cas9 gene-editing technology

β⁶⁵⁴-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 β⁶⁵⁴ mice. Gene-edited mice were generated by microinjection of sgRNA and Cas9 mRNA into one-cell embryos of β⁶⁵⁴ 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 β⁶⁵⁴ founders and their offspring were significantly improved compared to those of the non-edited β⁶⁵⁴ mice, consistent with the restoration of wild-type β-globin RNA expression. Notably, the survival rate of gene-edited heterozygous β⁶⁵⁴ mice increased significantly, and live-born homozygous β⁶⁵⁴ mice were observed. Our study demonstrated a new and effective gene-editing approach that may provide groundwork for the exploration of β⁶⁵⁴-thalassemia therapy in the future.