Disruption of splicing-regulatory elements using CRISPR/Cas9 to rescue spinal muscular atrophy in human iPSCs and mice


Li Jin-Jing12,Lin Xiang12,Tang Cheng3,Lu Ying-Qian1,Hu Xinde3,Zuo Erwei3,Li He3,Ying Wenqin3,Sun Yidi4,Lai Lu-Lu1,Chen Hai-Zhu1,Guo Xin-Xin1,Zhang Qi-Jie12,Wu Shuang1,Zhou Changyang3,Shen Xiaowen3,Wang Qifang3,Lin Min-Ting12,Ma Li-Xiang5,Wang Ning12,Krainer Adrian R6,Shi Linyu3,Yang Hui3,Chen Wan-Jin12


1. Department of Neurology and Institute of Neurology, The First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, China

2. Fujian Key Laboratory of Molecular Neurology, Fujian Medical University, Fuzhou 350005, China

3. Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

4. Key Lab of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

5. Department of Anatomy, Histology & Embryology, Shanghai Medical College, Fudan University, Shanghai 200032, China

6. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA


Abstract We here report a genome-editing strategy to correct spinal muscular atrophy (SMA). Rather than directly targeting the pathogenic exonic mutations, our strategy employed Cas9 and guide-sgRNA for the targeted disruption of intronic splicing-regulatory elements. We disrupted intronic splicing silencers (ISSs, including ISS-N1 and ISS + 100) of survival motor neuron (SMN) 2, a key modifier gene of SMA, to enhance exon 7 inclusion and full-length SMN expression in SMA iPSCs. Survival of splicing-corrected iPSC-derived motor neurons was rescued with SMN restoration. Furthermore, co-injection of Cas9 mRNA from Streptococcus pyogenes (SpCas9) or Cas9 from Staphylococcus aureus (SaCas9) alongside their corresponding sgRNAs targeting ISS-N1 into zygotes rescued 56% and 100% of severe SMA transgenic mice (Smn−/−, SMN2tg/−). The median survival of the resulting mice was extended to >400 days. Collectively, our study provides proof-of-principle for a new strategy to therapeutically intervene in SMA and other RNA-splicing-related diseases.


National Natural Science Foundation of China

Innovation of Science and Technology of Fujian Province

National Science and Technology major project

Shanghai City Committee of Science and Technology project


Oxford University Press (OUP)



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