Repurposing the CRISPR-Cas9 system for targeted DNA methylation.
Repurposing the CRISPR-Cas9 system for targeted DNA methylation.
复制标题
重新利用用于靶向DNA甲基化的CRISPR-CAS9系统。
DOI：
10.1093/nar/gkw159
复制
发表时间：
2016-07-08
影响因子：
14.9
通讯作者：
Zoldoš V
中科院分区：
文献类型：
--
作者：
Vojta A;Dobrinić P;Tadić V;Bočkor L;Korać P;Julg B;Klasić M;Zoldoš V
表观遗传学研究依赖于表观遗传标记和基因表达模式之间的相关性。为表观基因组编辑开发的技术现在能够直接研究精确的表观遗传修饰和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，已经在癌症治疗中用于重塑异常的表观遗传景观。然而，这是使用表观遗传抑制剂非选择性地实现的。在特定位点进行表观遗传编辑是一种新的方法，可以选择性地和遗传性地改变基因表达。在这里，我们开发了一种基于CRISPR-Cas9的特异性DNA甲基化工具，由失活的Cas9（dCas 9）核酸酶和DNA甲基转移酶DNMT 3A的催化结构域组成，通过将指导RNA共表达到任何20 bp DNA序列，然后是NGG三核苷酸。我们证明了融合蛋白在约35 bp宽的区域内的靶向CpG甲基化。我们还表明，多个指导RNA可以将dCas 9-DNMT 3A构建体靶向多个相邻位点，这使得启动子的大部分甲基化成为可能。DNA甲基化活性对靶区域具有特异性，并且在有丝分裂中具有遗传性。最后，我们证明了靶基因座IL 6ST和BACH 2的更宽启动子区域的定向DNA甲基化降低了它们的表达。
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.