McCallister Tristan X, Lim Colin K W, Singh Mayuri, Zhang Sijia, Ahsan Najah S, Terpstra William M, Xiong Alisha Y, Zeballos C M Alejandra, Powell Jackson E, Drnevich Jenny, Kang Yifei, Gaj Thomas
Department of Bioengineering, The Grainger College of Engineering, University of Illinois Urbana-Champaign, Urbana, IL, USA.
High-Performance Biological Computing, Roy J. Carver Biotechnology Center, University of Illinois Urbana-Champaign, Urbana, IL, USA.
Nat Commun. 2025 Jan 8;16(1):460. doi: 10.1038/s41467-024-55548-5.
An abnormal expansion of a GGGGCC (GC) hexanucleotide repeat in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two debilitating neurodegenerative disorders driven in part by gain-of-function mechanisms involving transcribed forms of the repeat expansion. By utilizing a Cas13 variant with reduced collateral effects, we develop here a high-fidelity RNA-targeting CRISPR-based system for C9ORF72-linked ALS/FTD. When delivered to the brain of a transgenic rodent model, this Cas13-based platform curbed the expression of the GC repeat-containing RNA without affecting normal C9ORF72 levels, which in turn decreased the formation of RNA foci, reduced the production of a dipeptide repeat protein, and reversed transcriptional deficits. This high-fidelity system possessed improved transcriptome-wide specificity compared to its native form and mediated targeting in motor neuron-like cells derived from a patient with ALS. These results lay the foundation for the implementation of RNA-targeting CRISPR technologies for C9ORF72-linked ALS/FTD.
C9ORF72基因中四碱基对GGGGCC（GC）六核苷酸重复序列的异常扩增是肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）最常见的遗传病因，这两种使人衰弱的神经退行性疾病部分由涉及重复扩增转录形式的功能获得机制驱动。通过利用一种具有降低的附带效应的Cas13变体，我们在此开发了一种用于C9ORF72相关ALS/FTD的高保真RNA靶向CRISPR系统。当将该系统递送至转基因啮齿动物模型的大脑时，这种基于Cas13的平台抑制了含GC重复序列的RNA的表达，而不影响正常的C9ORF72水平，这反过来又减少了RNA病灶的形成，降低了二肽重复蛋白的产生，并逆转了转录缺陷。与天然形式相比，这种高保真系统具有更高的全转录组特异性，并介导了对来自一名ALS患者的运动神经元样细胞的靶向作用。这些结果为将RNA靶向CRISPR技术应用于C9ORF72相关ALS/FTD奠定了基础。
