Spinal muscular atrophy (SMA) is a devastating neuromuscular disease caused by mutations in the survival motor neuron 1 (SMN1) gene. Gene editing technology repairs the conversion of the 6th base T to C in exon 7 of the paralogous SMN2 gene, compensating for the SMN protein expression and promoting the survival and function of motor neurons. However, low editing efficiency and unintended off‐target effects limit the application of this technology. Here, we optimized a TaC9‐adenine base editor (ABE) system by combining Cas9 nickase with the transcription activator‐like effector (TALE)‐adenosine deaminase fusion protein to effectively and precisely edit SMN2 without detectable Cas9 dependent off‐target effects in human cell lines. We also generated human SMA‐induced pluripotent stem cells (SMA‐iPSCs) through the mutation of the splice acceptor or deletion of the exon 7 of SMN1. TaC9‐R10 induced 45% SMN2 T6 > C conversion in the SMA‐iPSCs. The SMN2 T6 > C splice‐corrected SMA‐iPSCs were directionally differentiated into motor neurons, exhibiting SMN protein recovery and antiapoptosis ability. Therefore, the TaC9‐ABE system with dual guides from the combination of Cas9 with TALE could be a potential therapeutic strategy for SMA with high efficacy and safety.

中文翻译：

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TaC9-ABE 介导的人类 SMN2 基因校正的改进


脊髓性肌萎缩症 (SMA) 是一种毁灭性的神经肌肉疾病，由运动神经元存活基因 1 (SMN1) 基因突变引起。基因编辑技术修复旁系同源SMN2基因第7号外显子第6个碱基T至C的转换，补偿SMN蛋白表达，促进运动神经元的存活和功能。然而，低编辑效率和非预期的脱靶效应限制了该技术的应用。在这里，我们通过将 Cas9 切口酶与转录激活因子样效应器 (TALE)-腺苷脱氨酶融合蛋白相结合，优化了 TaC9-腺嘌呤碱基编辑器 (ABE) 系统，以有效、精确地编辑 SMN2，而不会在人体细胞中检测到 Cas9 依赖性脱靶效应线。我们还通过剪接受体的突变或 SMN1 外显子 7 的缺失产生了人类 SMA 诱导的多能干细胞 (SMA-iPSC)。 TaC9-R10 在 SMA-iPSC 中诱导 45% SMN2 T6 > C 转化。 SMN2 T6 > C 剪接校正的 SMA-iPSC 定向分化为运动神经元，表现出 SMN 蛋白恢复和抗凋亡能力。因此，Cas9与TALE组合的双引导TaC9-ABE系统可能是一种高效且安全的SMA潜在治疗策略。