BACKGROUND Recent advances in genome editing have facilitated the direct manipulation of not only the genome, but also the epigenome. Genome editing is typically performed by introducing a single CRISPR/Cas9-mediated double-strand break (DSB), followed by non-homologous end joining (NHEJ)- or homology-directed repair-mediated repair. Epigenome editing, and in particular methylation of CpG dinucleotides, can be performed using catalytically inactive Cas9 (dCas9) fused to a methyltransferase domain. However, for investigations of the role of methylation in gene silencing, studies based on dCas9-methyltransferase have limited resolution and are potentially confounded by the effects of binding of the fusion protein. As an alternative strategy for epigenome editing, we tested CRISPR/Cas9 dual cutting of the genome in the presence of in vitro methylated exogenous DNA, with the aim of driving replacement of the DNA sequence intervening the dual cuts via NHEJ. RESULTS In a proof of concept at the HPRT1 promoter, successful replacement events with heavily methylated alleles of a CpG island resulted in functional silencing of the HPRT1 gene. Although still limited in efficiency, our study demonstrates concurrent epigenome and genome editing in a single event. CONCLUSIONS This study opens the door to investigations of the functional consequences of methylation patterns at single CpG dinucleotide resolution. Our results furthermore support the conclusion that promoter methylation is sufficient to functionally silence gene expression.

中文翻译：

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通过 CRISPR 介导的序列替换进行同步基因组和表观基因组编辑。

背景技术基因组编辑的最新进展不仅促进了基因组的直接操作，而且还促进了表观基因组的直接操作。基因组编辑通常通过引入单个 CRISPR/Cas9 介导的双链断裂 (DSB)，然后进行非同源末端连接 (NHEJ) 或同源定向修复介导的修复来进行。表观基因组编辑，特别是 CpG 二核苷酸的甲基化，可以使用与甲基转移酶结构域融合的催化失活 Cas9 (dCas9) 进行。然而，对于甲基化在基因沉默中的作用的研究，基于 dCas9-甲基转移酶的研究分辨率有限，并且可能因融合蛋白结合的影响而混淆。作为表观基因组编辑的替代策略，我们在体外甲基化外源 DNA 存在的情况下测试了基因组的 CRISPR/Cas9 双切割，目的是通过 NHEJ 驱动干预双切割的 DNA 序列的替换。结果 在 HPRT1 启动子的概念验证中，CpG 岛重甲基化等位基因的成功替换事件导致 HPRT1 基因的功能性沉默。尽管效率仍然有限，但我们的研究证明了在单个事件中同时进行表观基因组和基因组编辑。结论 这项研究为研究单 CpG 二核苷酸分辨率的甲基化模式的功能后果打开了大门。我们的结果进一步支持启动子甲基化足以在功能上沉默基因表达的结论。