Vojta Aleksandar, Dobrinić Paula, Tadić Vanja, Bočkor Luka, Korać Petra, Julg Boris, Klasić Marija, Zoldoš Vlatka
Department of Biology, Division of Molecular Biology, University of Zagreb, Faculty of Science, Zagreb, HR-10000, Croatia.
Ragon Institute of MGHT, MIT and Harvard, 400 Technology Square, Cambridge, MA, USA.
Nucleic Acids Res. 2016 Jul 8;44(12):5615-28. doi: 10.1093/nar/gkw159. Epub 2016 Mar 11.
Epigenetic studies relied so far on correlations between epigenetic marks and gene expression pattern. Technologies developed for epigenome editing now enable direct study of functional relevance of precise epigenetic modifications and gene regulation. The reversible nature of epigenetic modifications, including DNA methylation, has been already exploited in cancer therapy for remodeling the aberrant epigenetic landscape. However, this was achieved non-selectively using epigenetic inhibitors. Epigenetic editing at specific loci represents a novel approach that might selectively and heritably alter gene expression. Here, we developed a CRISPR-Cas9-based tool for specific DNA methylation consisting of deactivated Cas9 (dCas9) nuclease and catalytic domain of the DNA methyltransferase DNMT3A targeted by co-expression of a guide RNA to any 20 bp DNA sequence followed by the NGG trinucleotide. We demonstrated targeted CpG methylation in a ∼35 bp wide region by the fusion protein. We also showed that multiple guide RNAs could target the dCas9-DNMT3A construct to multiple adjacent sites, which enabled methylation of a larger part of the promoter. DNA methylation activity was specific for the targeted region and heritable across mitotic divisions. Finally, we demonstrated that directed DNA methylation of a wider promoter region of the target loci IL6ST and BACH2 decreased their expression.
迄今为止，表观遗传学研究依赖于表观遗传标记与基因表达模式之间的相关性。目前开发的用于表观基因组编辑的技术能够直接研究精确表观遗传修饰与基因调控的功能相关性。表观遗传修饰的可逆性，包括DNA甲基化，已在癌症治疗中用于重塑异常的表观遗传格局。然而，这是通过使用表观遗传抑制剂非选择性地实现的。特定基因座的表观遗传编辑代表了一种可能选择性地和可遗传地改变基因表达的新方法。在这里，我们开发了一种基于CRISPR-Cas9的工具用于特定的DNA甲基化，该工具由失活的Cas9（dCas9）核酸酶和DNA甲基转移酶DNMT3A的催化结构域组成，通过将引导RNA共表达靶向任何20bp的DNA序列，随后是NGG三核苷酸。我们通过融合蛋白在约35bp宽的区域内证明了靶向的CpG甲基化。我们还表明，多个引导RNA可以将dCas9-DNMT3A构建体靶向多个相邻位点，从而使启动子的更大区域发生甲基化。DNA甲基化活性对靶向区域具有特异性，并且在有丝分裂过程中是可遗传的。最后，我们证明了目标基因座IL6ST和BACH2的更宽启动子区域的定向DNA甲基化降低了它们的表达。
