Transcription of expanded microsatellite repeats is associated with multiple human diseases, including myotonic dystrophy, Fuchs endothelial corneal dystrophy, and C9orf72-ALS/FTD. Reducing production of RNA and proteins arising from these expanded loci holds therapeutic benefit. Here, we tested the hypothesis that deactivated Cas9 enzyme impedes transcription across expanded microsatellites. We observed a repeat length-, PAM-, and strand-dependent reduction of repeat-containing RNAs upon targeting dCas9 directly to repeat sequences; targeting the non-template strand was more effective. Aberrant splicing patterns were rescued in DM1 cells, and production of RAN peptides characteristic of DM1, DM2, and C9orf72-ALS/FTD cells was drastically decreased. Systemic delivery of dCas9/gRNA by adeno-associated virus led to reductions in pathological RNA foci, rescue of chloride channel 1 protein expression, and decreased myotonia. These observations suggest that transcription of microsatellite repeat-containing RNAs is more sensitive to perturbation than transcription of other RNAs, indicating potentially viable strategies for therapeutic intervention.

扩展的微卫星重复序列的转录与多种人类疾病有关，包括强直性肌营养不良，Fuchs内皮角膜营养不良和C9orf72 -ALS / FTD。减少由这些扩增的基因座产生的RNA和蛋白质的产生具有治疗益处。在这里，我们测试了停用Cas9酶会阻止扩展的微卫星转录的假说。我们观察到直接将dCas9靶向重复序列后，包含重复序列的RNA的重复长度，PAM和链依赖的减少。靶向非模板链更为有效。在DM1细胞中拯救了异常的剪接模式，并产生了DM1，DM2和C9orf72特有的RAN肽-ALS / FTD细胞急剧减少。腺相关病毒对dCas9 / gRNA的全身递送导致病理性RNA病灶减少，氯通道1蛋白表达得以挽救并减少了肌强直。这些观察结果表明，与其他RNA的转录相比，含微卫星重复序列的RNA的转录对扰动更为敏感，这表明可能存在可行的治疗干预策略。