Editing DNA Methylation in the Mammalian Genome

Abstract

SUMMARY Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice demonstrating their wide utility for functional studies of epigenetic regulation.

Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain

“…18,19,21,22 TET enzymes mediate DNA demethylation indirectly through oxidation of 5mC to 5hmC, and further oxidation of 5hmC to 5fC and 5caC. 29,30 However, bisulfite sequencing does not distinguish between unmethylated C and either 5fC or 5caC, since these two derivatives are not resistant to deamination.…”

“…16,17 Recent studies have shown targeted DNA demethylation and gene reactivation in mammalian cells by fusing different enzymes involved in DNA demethylation to diverse DBDs. [18][19][20][21][22] However, no efforts have been reported so far to express or target plant 5mC DNA glycosylases, which directly excise 5mC from DNA.…”

CRISPR-based reagents to study the influence of the epigenome on gene expression

“…2b). Similarly, fusion of histone or DNA methyl transferases to Cas9 has been used to prove the influence of specific DNA methylation events 61, 62, 63, 64, 65, 66.…”

CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes

“…Tethering the catalytic domain of the DNA demethylase Tet1 to dCas9 has been used for targeted DNA demethylation (Figure 4a). These fusions can induce over 90% demethylation of CpG islands within a 200-bp range of the target site Morita et al, 2016;Liu et al, 2016). Researchers have also developed a histone-modifying CRISPR-based tool in which the catalytic domain of human acetyltransferase p300 was fused to the C-terminus of dCas9.…”