面肩肱型肌营养不良症（FSHD）是常见的成人肌营养不良症，无有效治疗。基因定位于4q35区域，为大卫星序列（D4Z4）的多拷贝缺失，引发D4Z4特异性甲基化水平下降，导致其内部DUX4基因上调表达，产生细胞毒作用。由于具体甲基化调控通路不清，限制了精准的干预。我们前期证实FSHD患者存在共同致病基因型SSLP161-D4Z4/PAS-4qA，通过靶向甲基化检测，发现4q35-D4Z4甲基化水平越低表型越重。为明确甲基化调控通路，应用scRNA-seq、Hi-C、三代测序等技术筛选甲基化修饰相关基因，探讨DUX4基因上调表达机制；同时，为探究定点甲基化修复是否起到有效干预，构建dCas9-SunTag-Dnmt3a表达系统，转染FSHD-iPSCs并转分化成肌细胞，包装AVV病毒，注射成体FLExDUX4小鼠，靶向提高D4Z4的甲基化水平，明确其对DUX4抑制效应及表型变化，为治疗提供线索。

acioscapulohumeral muscular dystrophy (FSHD) is the third most common type of muscular dystrophy without effective treatment. FSHD is caused by a decrease in the copy number of polymorphic D4Z4 repeats (DRs) locating in the region of chromosome 4q35. Multiple variation resulted in DNA hypomethylation has been confirmed, as a consequence, the inner DUX4 gene was overexpression in skeletal muscles and consequently led to the “gain of toxic function”. Precise intervention treatment was limited due to the unclear mechanism concerning the regulation of specific methylation. We had been identified that FSHD carried the pathogenic genotype of SSLP161-D4Z4/PAS-4qA in previous studies. Targeted pyrosequencing displayed that a lower methylation level of D4Z4 associating with a severer clinical phenotype. In order to investigate the specific methylation regulation, single cell RNA-sequencing, Hi-C and third-generation sequencing would be used to screen the related genes regulating methylation and to explore the mechanism about the overexpression of DUX4 gene. In addition, dCas9-Dnmt3a-single guide RNA (sgRNA) would be transferred to patient-derived iPSCs to explore the potential therapeutic strategies of targeted demethylation. We further would inject the AAV-dCas9-Dnmt3a-sgRNA into the muscles of DUX4-fl mice to restore the D4Z4 methylation level, to suppress the expression of toxic DUX4 gene and to rescue the clinical phenotype. We would provide clues that DNA methylation editing of the 4q35 D4Z4 is sufficient for FSHD therapy.