A significant number of X-linked genes escape from X chromosome inactivation and are associated with a distinct epigenetic signature. One epigenetic modification that strongly correlates with X-escape is reduced DNA methylation in promoter regions. Here, we created an artificial escape by editing DNA methylation on the promoter of CDKL5, a gene causative for an infantile epilepsy, from the silenced X-chromosomal allele in human neuronal-like cells. We identify that a fusion of the catalytic domain of TET1 to dCas9 targeted to the CDKL5 promoter using three guide RNAs causes significant reactivation of the inactive allele in combination with removal of methyl groups from CpG dinucleotides. Strikingly, we demonstrate that co-expression of TET1 and a VP64 transactivator have a synergistic effect on the reactivation of the inactive allele to levels >60% of the active allele. We further used a multi-omics assessment to determine potential off-targets on the transcriptome and methylome. We find that synergistic delivery of dCas9 effectors is highly selective for the target site. Our findings further elucidate a causal role for reduced DNA methylation associated with escape from X chromosome inactivation. Understanding the epigenetics associated with escape from X chromosome inactivation has potential for those suffering from X-linked disorders.

中文翻译：

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通过 CDKL5 基因的 DNA 甲基化编辑从 XCI 中人工逃逸。

大量 X 连锁基因从 X 染色体失活中逃脱，并与独特的表观遗传特征相关。一种与 X 逃逸密切相关的表观遗传修饰是启动子区域的 DNA 甲基化减少。在这里，我们通过编辑 CDKL5 启动子上的 DNA 甲基化来人工逃逸，CDKL5 是一种婴儿癫痫的致病基因，来自人类神经元样细胞中沉默的 X 染色体等位基因。我们发现，使用三个引导 RNA 将 TET1 的催化域融合到靶向 CDKL5 启动子的 dCas9 会导致失活等位基因显着重新激活，同时从 CpG 二核苷酸中去除甲基。引人注目的是，我们证明 TET1 和 VP64 反式激活因子的共表达对将非活性等位基因重新激活至 > 60% 活性等位基因的水平具有协同作用。我们进一步使用多组学评估来确定转录组和甲基化组的潜在脱靶。我们发现 dCas9 效应子的协同传递对目标站点具有高度选择性。我们的研究结果进一步阐明了与逃避 X 染色体失活相关的 DNA 甲基化减少的因果关系。了解与逃避 X 染色体失活相关的表观遗传学对于那些患有 X 连锁疾病的人来说具有潜力。我们发现 dCas9 效应子的协同传递对目标站点具有高度选择性。我们的研究结果进一步阐明了与逃避 X 染色体失活相关的 DNA 甲基化减少的因果关系。了解与逃避 X 染色体失活相关的表观遗传学对于那些患有 X 连锁疾病的人来说具有潜力。我们发现 dCas9 效应子的协同传递对目标站点具有高度选择性。我们的研究结果进一步阐明了与逃避 X 染色体失活相关的 DNA 甲基化减少的因果关系。了解与逃避 X 染色体失活相关的表观遗传学对于那些患有 X 连锁疾病的人来说具有潜力。