Myotonic dystrophy type 1 is the most common type of adult-onset muscular dystrophy. This is an autosomal dominant disorder and caused by the expansion of the CTG repeat in the 3′ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Messenger RNAs containing these expanded repeats form aggregates as nuclear RNA foci. Then, RNA binding proteins, including muscleblind-like 1, are sequestered to the RNA foci, leading to systemic abnormal RNA splicing. In this study, we used CRISPR-Cas9 genome editing to excise this CTG repeat. Dual cleavage at the 5′ and 3′ regions of the repeat using a conventional Cas9 nuclease and a double nicking with Cas9 nickase successfully excised the CTG repeat. Subsequently, the formation of the RNA foci was markedly reduced in patient-derived fibroblasts. However, contrary to expectations, a considerable amount of off-target digestions and on-target genomic rearrangements were observed using high-throughput genome-wide translocation sequencing. Finally, the suppression of DMPK transcripts using CRISPR interference significantly decreased the intensity of RNA foci. Our results indicate that close attention should be paid to the unintended mutations when double-strand breaks are generated by CRISPR-Cas9 for therapeutic purposes. Alternative approaches independent of double-strand breaks, including CRISPR interference, may be considered.

1型强直性肌营养不良是成人发病的肌肉营养不良的最常见类型。这是常染色体显性遗传疾病，由肌营养不良症肌强直蛋白激酶（DMPK） 3'非翻译区中CTG重复序列的扩增引起）基因。包含这些扩展重复序列的信使RNA形成聚集体，成为核RNA焦点。然后，将包括肌肉盲样1在内的RNA结合蛋白隔离到RNA病灶中，导致全身性异常RNA剪接。在这项研究中，我们使用CRISPR-Cas9基因组编辑来切除该CTG重复序列。使用常规Cas9核酸酶在重复序列的5'和3'区域进行双重切割，并用Cas9切口酶进行双切口成功切除了CTG重复序列。随后，在患者来源的成纤维细胞中，RNA病灶的形成明显减少。然而，与预期相反，使用高通量全基因组易位测序观察到大量脱靶消化和脱靶基因组重排。最后，抑制DMPK使用CRISPR干扰的转录本可显着降低RNA灶的强度。我们的结果表明，当CRISPR-Cas9产生双链断裂用于治疗目的时，应密切注意意外突变。可以考虑独立于双链断裂的替代方法，包括CRISPR干扰。