The G4C2 hexanucleotide repeat expansion (HRE) in C9orf72 is the commonest cause of familial amyotrophic lateral sclerosis (ALS). A number of different methods have been used to generate isogenic control lines using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and non-homologous end-joining by deleting the repeat region, with the risk of creating indels and genomic instability. In this study, we demonstrate complete correction of an induced pluripotent stem cell (iPSC) line derived from a C9orf72-HRE positive ALS/frontotemporal dementia patient using CRISPR/Cas9 genome editing and homology-directed repair (HDR), resulting in replacement of the excised region with a donor template carrying the wild-type repeat size to maintain the genetic architecture of the locus. The isogenic correction of the C9orf72 HRE restored normal gene expression and methylation at the C9orf72 locus, reduced intron retention in the edited lines and abolished pathological phenotypes associated with the C9orf72 HRE expansion in iPSC-derived motor neurons (iPSMNs). RNA sequencing of the mutant line identified 2220 differentially expressed genes compared with its isogenic control. Enrichment analysis demonstrated an over-representation of ALS relevant pathways, including calcium ion dependent exocytosis, synaptic transport and the Kyoto Encyclopedia of Genes and Genomes ALS pathway, as well as new targets of potential relevance to ALS pathophysiology. Complete correction of the C9orf72 HRE in iPSMNs by CRISPR/Cas9-mediated HDR provides an ideal model to study the earliest effects of the hexanucleotide expansion on cellular homeostasis and the key pathways implicated in ALS pathophysiology.

中文翻译：

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通过使用同源定向修复的 CRISPR/Cas9 基因组编辑，在 C9orf72 中携带六核苷酸扩增突变的诱导多能干细胞衍生运动神经元中肌萎缩侧索硬化相关表型的校正。

C9orf72中的 G4C2 六核苷酸重复扩增 (HRE)是家族性肌萎缩侧索硬化 (ALS) 的最常见原因。许多不同的方法已被用于生成等基因控制线，这些方法使用成簇的规则间隔短回文重复 (CRISPR)/Cas9 和通过删除重复区域的非同源末端连接，具有产生插入缺失和基因组不稳定性的风险。在这项研究中，我们展示了对源自C9orf72的诱导多能干细胞 (iPSC) 系的完全校正-HRE 阳性 ALS/额颞叶痴呆患者使用 CRISPR/Cas9 基因组编辑和同源定向修复 (HDR)，导致切除区域替换为携带野生型重复大小的供体模板，以维持基因座的遗传结构。C9orf72 HRE的同基因校正恢复了C9orf72基因座的正常基因表达和甲基化，减少了编辑系中的内含子保留，并消除了与C9orf72相关的病理表型iPSC 衍生运动神经元 (iPSMN) 中的 HRE 扩展。与其同基因对照相比，突变系的 RNA 测序鉴定了 2220 个差异表达基因。富集分析表明 ALS 相关通路的过度表现，包括钙离子依赖性胞吐作用、突触转运和京都基因和基因组百科全书 ALS 通路，以及与 ALS 病理生理学潜在相关的新靶点。通过 CRISPR/Cas9 介导的 HDR完全校正 iPSMN 中的C9orf72 HRE 为研究六核苷酸扩增对细胞稳态的最早影响以及与 ALS 病理生理学有关的关键途径提供了理想的模型。