Correction of RNA splicing defect in β<sup>654</sup>-thalassemia mice using CRISPR/Cas9 gene-editing technology
Dan Lu,
Xiuli Gong,
Yudan Fang,
Xinbing Guo,
Yanwen Chen,
Fan Yang,
Guijun Zhao,
Qingwen Ma,
Yitao Zeng,
Fanyi Zeng
Affiliations
Dan Lu
Shanghai Institute of Medical Genetics, Shanghai Children’s Hospital, Shanghai Jiao Tong University, Shanghai 200040
Xiuli Gong
Shanghai Institute of Medical Genetics, Shanghai Children’s Hospital, Shanghai Jiao Tong University, Shanghai 200040
Yudan Fang
Shanghai Institute of Medical Genetics, Shanghai Children’s Hospital, Shanghai Jiao Tong University, Shanghai 200040
Xinbing Guo
Shanghai Institute of Medical Genetics, Shanghai Children’s Hospital, Shanghai Jiao Tong University, Shanghai 200040
Yanwen Chen
Shanghai Institute of Medical Genetics, Shanghai Children’s Hospital, Shanghai Jiao Tong University, Shanghai 200040
Fan Yang
Shanghai Institute of Medical Genetics, Shanghai Children’s Hospital, Shanghai Jiao Tong University, Shanghai 200040
Guijun Zhao
Shanghai Institute of Medical Genetics, Shanghai Children’s Hospital, Shanghai Jiao Tong University, Shanghai 200040
Qingwen Ma
Shanghai Institute of Medical Genetics, Shanghai Children’s Hospital, Shanghai Jiao Tong University, Shanghai 200040
Yitao Zeng
Shanghai Institute of Medical Genetics, Shanghai Children’s Hospital, Shanghai Jiao Tong University, Shanghai 200040
Fanyi Zeng
Shanghai Institute of Medical Genetics, Shanghai Children’s Hospital, Shanghai Jiao Tong University, Shanghai 200040, China; Department of Histo-Embryology, Genetics and Developmental Biology, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China; Key Laboratory of Embryo Molecular Biology, Ministry of Health and Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai 200040
β654-thalassemia is a prominent Chinese subtype of b-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 b-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 b-globin RNA expression. Notably, the survival rate of gene-edited heterozygous β654 mice increased significantly, and liveborn 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.
