Department of Genome Regulation, Max Planck Institute for Molecular Genetics, 14195, Berlin, Germany.
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, 02138, USA.
Nat Commun. 2018 Feb 9;9(1):597. doi: 10.1038/s41467-017-02708-5.
In normal mammalian development cytosine methylation is essential and is directed to specific regions of the genome. Despite notable advances through mapping its genome-wide distribution, studying the direct contribution of DNA methylation to gene and genome regulation has been limited by the lack of tools for its precise manipulation. Thus, combining the targeting capability of the CRISPR-Cas9 system with an epigenetic modifier has attracted interest in the scientific community. In contrast to profiling the genome-wide cleavage of a nuclease competent Cas9, tracing the global activity of a dead Cas9 (dCas9) methyltransferase fusion protein is challenging within a highly methylated genome. Here, we report the generation and use of an engineered, methylation depleted but maintenance competent mouse ES cell line and find surprisingly ubiquitous nuclear activity of dCas9-methyltransferases. Subsequent experiments in human somatic cells refine these observations and point to an important difference between genetic and epigenetic editing tools that require unique experimental considerations.
在正常的哺乳动物发育过程中，胞嘧啶甲基化是必不可少的，并且针对基因组的特定区域。尽管通过绘制其全基因组分布取得了显著进展，但由于缺乏精确操纵 DNA 甲基化的工具，研究 DNA 甲基化对基因和基因组调控的直接贡献受到了限制。因此，将 CRISPR-Cas9 系统的靶向能力与表观遗传修饰剂结合使用，引起了科学界的兴趣。与分析能够切割核酸酶的 Cas9 在全基因组范围内的切割情况相比，在高度甲基化的基因组中追踪无活性 Cas9(dCas9)甲基转移酶融合蛋白的全局活性具有挑战性。在这里，我们报告了一种经过工程改造的、去甲基化但维持能力强的小鼠胚胎干细胞系的产生和应用，并令人惊讶地发现 dCas9-甲基转移酶在细胞核中普遍存在活性。随后在人体细胞中的实验进一步证实了这些观察结果，并指出了遗传编辑工具和表观遗传编辑工具之间的一个重要区别，这需要独特的实验考虑。
