Rewriting of the epigenome has risen as a promising alternative to gene editing for precision medicine. In nature, epigenetic silencing can result in complete attenuation of target gene expression over multiple mitotic divisions. However, persistent repression has been difficult to achieve in a predictable manner using targeted systems. Here, we report that persistent epigenetic memory required both a DNA methyltransferase (DNMT3A-dCas9) and a histone methyltransferase (Ezh2-dCas9 or KRAB-dCas9). We demonstrate that the histone methyltransferase requirement can be locus specific. Co-targeting Ezh2-dCas9, but not KRAB-dCas9, with DNMT3A-dCas9 and DNMT3L induced long-term HER2 repression over at least 50 days (approximately 57 cell divisions) and triggered an epigenetic switch to a heterochromatic environment. An increase in H3K27 trimethylation and DNA methylation was stably maintained and accompanied by a sustained loss of H3K27 acetylation. Interestingly, substitution of Ezh2-dCas9 with KRAB-dCas9 enabled long-term repression at some target genes (e.g., SNURF) but not at HER2, at which H3K9me3 and DNA methylation were transiently acquired and subsequently lost. Off-target DNA hypermethylation occurred at many individual CpG sites but rarely at multiple CpGs in a single promoter, consistent with no detectable effect on transcription at the off-target loci tested. Conversely, robust hypermethylation was observed at HER2. We further demonstrated that Ezh2-dCas9 required full-length DNMT3L for maximal activity and that co-targeting DNMT3L was sufficient for persistent repression by Ezh2-dCas9 or KRAB-dCas9. These data demonstrate that targeting different combinations of histone and DNA methyltransferases is required to achieve maximal repression at different loci. Fine-tuning of targeting tools is a necessity to engineer epigenetic memory at any given locus in any given cell type.

中文翻译：

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Ezh2-dCas9 和 KRAB-dCas9 能够以上下文相关的方式对表观遗传记忆进行工程设计。


表观基因组的重写已成为精准医学基因编辑的一种有前景的替代方案。在自然界中，表观遗传沉默可以导致多次有丝分裂中靶基因表达的完全减弱。然而，使用有针对性的系统很难以可预测的方式实现持续镇压。在这里，我们报告持久的表观遗传记忆需要 DNA 甲基转移酶 (DNMT3A-dCas9) 和组蛋白甲基转移酶 (Ezh2-dCas9 或 KRAB-dCas9)。我们证明组蛋白甲基转移酶的要求可以是位点特异性的。与 DNMT3A-dCas9 和 DNMT3L 共同靶向 Ezh2-dCas9（而非 KRAB-dCas9）诱导了至少 50 天的长期 HER2 抑制（大约 57 次细胞分裂），并触发了向异色环境的表观遗传转换。 H3K27 三甲基化和 DNA 甲基化的增加稳定维持，并伴随着 H3K27 乙酰化的持续丧失。有趣的是，用 KRAB-dCas9 替换 Ezh2-dCas9 可以对某些靶基因（例如 SNURF）进行长期抑制，但不能对 HER2 进行长期抑制，在 HER2 上，H3K9me3 和 DNA 甲基化是短暂获得的，随后又丢失。脱靶 DNA 高甲基化发生在许多单独的 CpG 位点，但很少发生在单个启动子中的多个 CpG，这与测试的脱靶位点对转录没有可检测到的影响一致。相反，在 HER2 处观察到强烈的高甲基化。我们进一步证明，Ezh2-dCas9 需要全长 DNMT3L 才能发挥最大活性，并且共靶向 DNMT3L 足以实现 Ezh2-dCas9 或 KRAB-dCas9 的持续抑制。这些数据表明，需要靶向组蛋白和 DNA 甲基转移酶的不同组合才能在不同位点实现最大抑制。 靶向工具的微调是在任何给定细胞类型的任何给定位点设计表观遗传记忆的必要条件。