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

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.