Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, and.
Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, Maryland, USA.
J Clin Invest. 2024 Sep 17;134(21):e179016. doi: 10.1172/JCI179016.
A hexanucleotide GGGGCC repeat expansion in the non-coding region of the C9orf72 gene is the most common genetic mutation identified in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The resulting repeat RNA and dipeptide repeat proteins from non-conventional repeat translation have been recognized as important markers associated with the diseases. CRISPR/Cas13d, a powerful RNA-targeting tool, has faced challenges in effectively targeting RNA with stable secondary structures. Here we report that CRISPR/Cas13d can be optimized to specifically target GGGGCC repeat RNA. Our results demonstrate that the CRISPR/Cas13d system can be harnessed to significantly diminish the translation of poly-dipeptides originating from the GGGGCC repeat RNA. This efficacy has been validated in various cell types, including induced pluripotent stem cells and differentiated motor neurons originating from C9orf72-ALS patients, as well as in C9orf72 repeat transgenic mice. These findings demonstrate the application of CRISPR/Cas13d in targeting RNA with intricate higher-order structures and suggest a potential therapeutic approach for ALS and FTD.
在 C9orf72 基因的非编码区域发现的六核苷酸 GGGGCC 重复扩展是肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）患者中最常见的遗传突变。非传统重复翻译产生的重复 RNA 和二肽重复蛋白已被认为是与疾病相关的重要标志物。CRISPR/Cas13d 是一种强大的 RNA 靶向工具，但在有效靶向具有稳定二级结构的 RNA 方面面临挑战。在这里，我们报告说，CRISPR/Cas13d 可以被优化为专门针对 GGGGCC 重复 RNA。我们的结果表明，可以利用 CRISPR/Cas13d 系统显著减少源自 GGGGCC 重复 RNA 的多二肽的翻译。这种功效已在各种细胞类型中得到验证，包括源自 C9orf72-ALS 患者的诱导多能干细胞和分化的运动神经元，以及 C9orf72 重复转基因小鼠。这些发现证明了 CRISPR/Cas13d 在靶向具有复杂高级结构的 RNA 方面的应用，并为 ALS 和 FTD 提供了一种潜在的治疗方法。
