Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, 21218, USA.
Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA.
Sci Rep. 2017 Jul 27;7(1):6732. doi: 10.1038/s41598-017-06757-0.
Mammalian genomes exhibit complex patterns of gene expression regulated, in part, by DNA methylation. The advent of engineered DNA methyltransferases (MTases) to target DNA methylation to specific sites in the genome will accelerate many areas of biological research. However, targeted MTases require clear design rules to direct site-specific DNA methylation and minimize the unintended effects of off-target DNA methylation. Here we report a targeted MTase composed of an artificially split CpG MTase (sMTase) with one fragment fused to a catalytically-inactive Cas9 (dCas9) that directs the functional assembly of sMTase fragments at the targeted CpG site. We precisely map RNA-programmed DNA methylation to targeted CpG sites as a function of distance and orientation from the protospacer adjacent motif (PAM). Expression of the dCas9-sMTase in mammalian cells led to predictable and efficient (up to ~70%) DNA methylation at targeted sites. Multiplexing sgRNAs enabled targeting methylation to multiple sites in a single promoter and to multiple sites in multiple promoters. This programmable de novo MTase tool might be used for studying mechanisms of initiation, spreading and inheritance of DNA methylation, and for therapeutic gene silencing.
哺乳动物基因组表现出复杂的基因表达调控模式，部分受 DNA 甲基化调控。工程化的 DNA 甲基转移酶（MTases）将 DNA 甲基化靶向基因组中的特定位点，将加速许多生物学研究领域的发展。然而，靶向 MTases 需要明确的设计规则来指导特异性 DNA 甲基化，并最小化非靶向 DNA 甲基化的意外影响。在这里，我们报告了一种由人工分割的 CpG MTase（sMTase）与一个融合了无催化活性 Cas9（dCas9）的片段组成的靶向 MTase，该片段指导 sMTase 片段在靶向 CpG 位点的功能组装。我们精确地将 RNA 编程的 DNA 甲基化映射到靶向 CpG 位点，作为距离和取向从原间隔基序（PAM）的函数。在哺乳动物细胞中表达 dCas9-sMTase 导致靶向位点的 DNA 甲基化可预测且高效（高达约 70%）。多重 sgRNAs 使靶向单个启动子中的多个位点和多个启动子中的多个位点的甲基化成为可能。这种可编程的从头 MTase 工具可用于研究 DNA 甲基化的起始、扩散和遗传机制，以及用于治疗性基因沉默。
