CRISPR/Cas9介导的FMR1基因激活策略优化

FMR1基因5’端非编码区CGG重复序列异常扩增与启动子区高甲基化是导致脆性X综合征（FXS）的分子机制，寻找激活FMR1的治疗策略一直是该领域的难题。前期研究中，我们利用CRISPR/Cas9基因编辑技术在多个FXS细胞模型中精确切除了全突变CGG重复序列，剪切后20%～67%细胞克隆的FMR1在转录与翻译水平被持续、显著的激活，且激活克隆的FMR1启动子区均处于低甲基化状态，未激活克隆的FMR1启动子区均处于高甲基化状态，提示切除全突变CGG重复序列可以通过诱导启动子区DNA去甲基化以激活FMR1但激活效率并非100%。本课题拟以FXS-iPSC为模型，利用CRISPR/Cas9基因编辑技术，通过细胞周期特异性剪切、剪切全突变序列并诱导启动子区去甲基化、替换全突变序列为正常重复序列，探究更为优化的FMR1基因激活策略。

Fragile X syndrome (FXS) is a common cause of intellectual disability that is most often due to a CGG-repeat expansion mutation in the FMR1 gene that triggers epigenetic gene silencing. How to reactivate FMR1 remains a key unanswered question. We excised the expanded CGG-repeat in both somatic cell hybrids containing the human fragile X chromosome and human FXS iPS cells using the CRISPR/Cas9 genome editing. Transcriptional reactivation was observed in approximately 67% of the CRISPR cut hybrid colonies and in 20% of isolated human FXS iPSC colonies. The reactivated cells produced FMRP and exhibited a decline in DNA methylation at the FMR1 locus. These data demonstrate the excision of the expanded CGG-repeat from the fragile X chromosome can result in FMR1 reactivation by inducing promoter demethylation. However, the reactivation rate is not 100%. We aim to further optimize the FMR1 reactivation strategy in a human FXS iPSC model, from perspectives of cell cycle-specific cleavage, CGG excision plus promoter demethylation, and substitution of full mutation with normal repeats. Outcome of this research will provide insight into the exploration of the FXS therapeutic strategy.