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^−/−, SMN2^tg/−). 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.

中文翻译：

---

使用 CRISPR/Cas9 破坏剪接调节元件以挽救人类 iPSC 和小鼠的脊髓性肌萎缩症

我们在这里报告了一种纠正脊髓性肌萎缩症 (SMA) 的基因组编辑策略。我们的策略不是直接针对致病性外显子突变，而是采用 Cas9 和 guide-sgRNA 来靶向破坏内含子剪接调控元件。我们破坏了运动神经元存活 (SMN) 2 的内含子剪接消音器（ISS，包括 ISS-N1 和 ISS + 100），这是 SMA 的一个关键修饰基因，以增强 SMA iPSC 中的外显子 7 包含和全长 SMN 表达。通过 SMN 修复挽救了剪接校正后的 iPSC 衍生运动神经元的存活。此外，共注射来自化脓性链球菌(SpCas9) 的 Cas9 mRNA 或来自金黄色葡萄球菌的 Cas9(SaCas9) 及其相应的 sgRNA 将 ISS-N1 靶向受精卵，拯救了 56% 和 100% 的严重 SMA 转基因小鼠（Smn ^-/-，SMN2 ^tg/-）。所得小鼠的中位存活期延长至 >400 天。总的来说，我们的研究为治疗干预 SMA 和其他 RNA 剪接相关疾病的新策略提供了原理证明。