Targeted DNA methylation in human cells using engineered dCas9-methyltransferases

Abstract

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.

CRISPR/Cas9-Based Engineering of the Epigenome

“…More recently several groups have engineered dCas9 fused to the catalytic domain of distinct DNMTs to methylate CpG islands, CTCF binding sites, and promoter and enhancer regions in cancer cell lines, ESCs and primary cells (Liu et al, 2016; McDonald et al, 2016; Vojta et al, 2016; Lei et al, 2017; Xiong et al, 2017) (Fig. 1C).…”

Concurrent Genome and Epigenome Editing by CRISPR-Mediated Sequence Replacement

“…Finally, an important limitation of our approach is the effectively low efficiency of methylation. This study showed much lower methylation rates (<1%) as compared to the dCas9methyltransferase fusion protein approach (30-70%) (Liu et al 2016;Amabile et al 2016;Vojta et al 2016;McDonald et al 2016;Stepper et al 2017;Lei et al 2017;Xiong et al 2017). This is due to a combination of factors, including the low transfection efficiency and rate of NHEJ of the Hap1 cell line, the lower integration rate of methylated DNA, and the availability of alternative outcomes that are also selected for, e.g.…”

“…For targeted methylation, this CRISPR/NHEJ approach represents an alternative to the previously demonstrated dCas9-methyltransferase domain fusion protein approach (Liu et al 2016;Amabile et al 2016;Vojta et al 2016;McDonald et al 2016;Stepper et al 2017;Lei et al 2017;Xiong et al 2017). While both approaches can produce targeted, scarless methylation of genomic DNA, the CRISPR/NHEJ approach is distinguished by the potential to program precisely which subsets of CpG dinucleotides are methylated, e.g.…”

“…A second, recently demonstrated approach uses a catalytically inactive Cas9 as a targeting domain fused to a DNA methyltransferase domain for methylation of CpG dinucleotides (Liu et al 2016;Amabile et al 2016;Vojta et al 2016;McDonald et al 2016;Stepper et al 2017;Lei et al 2017;Xiong et al 2017;Adli 2018). This approach has lower efficiency and results in methylation of multiple CpGs surrounding the target site, requiring multiple guides if the goal is to methylate a region.…”

Harnessing natural DNA modifying activities for editing of the genome and epigenome

“…A recent study using a split methyltransferase fused to dCas9 exhibited high specificity to target sites and suggested that the strategy limited off-target effects [53]. Although not yet attached to a targeting scaffold, TET2 has also been split and placed under the control of a small molecule modulator: Two inactive fragments were individually fused with either FKBP12 or FRB, and upon the addition of rapamycin, TET2 reassembled into a functional enzyme [54].…”