Abstract
Despite the increased interest in epigenetic research, its progress has been hampered by a lack of satisfactory tools to control epigenetic factors in specific genomic regions. Until now, many attempts to manipulate DNA methylation have been made using drugs but these drugs are not target-specific and have global effects on the whole genome. However, due to new genome editing technologies, potential epigenetic factors can now possibly be regulated in a site-specific manner. Here, we demonstrate the utility of CRISPR/Cas9 to modulate methylation at specific CpG sites and to elicit gene expression. We targeted the murine Oct4 gene which is transcriptionally locked due to hypermethylation at the promoter region in NIH3T3 cells. To induce site-specific demethylation at the Oct4 promoter region and its gene expression, we used the CRISPR/Cas9 knock-in and CRISPR/dCas9-Tet1 systems. Using these two approaches, we induced site-specific demethylation at the Oct4 promoter and confirmed the up-regulation of Oct4 expression. Furthermore, we confirmed that the synergistic effect of DNA demethylation and other epigenetic regulations increased the expression of Oct4 significantly. Based on our research, we suggest that our proven epigenetic editing methods can selectively modulate epigenetic factors such as DNA methylation and have promise for various applications in epigenetics.
Highlights
Epigenetics is the study of the heritable changes in gene expression without altering the underlying DNA sequences[1,2,3]
In the NCBI database (NCBI Reference Sequence: NC_000083.6), it is known that there are 16 CpG sites in this region; in the NIH3T3 cells used for our experiments, the cytosine at the −420 site was substituted with thymine, resulting in a total of 15 CpGs
The cytosine at the −110 site was thoroughly unmethylated as reported in a previous study[27]; this site was excluded from our target list
Summary
Epigenetics is the study of the heritable changes in gene expression without altering the underlying DNA sequences[1,2,3]. To use the CRISPR/Cas[9] system in an epigenetic study, we developed two strategies to regulate the demethylation of the Oct[4] promoter and to induce the expression of the Oct[4] gene in differentiated cells. To avoid unknown side-effects caused by the mutated sequence on the promoter, we applied a CRISPR/dCas9-based demethylation system[29] and made an effort to improve this system by inducing chromatin remodeling To this end, we used a catalytically inactivated dCas[9] fused to the catalytic domain of Ten-Eleven Translocation dioxygenase 1 (Tet1) which catalyzes the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC)[30,31]. The aim of this research was to investigate a potential selective demethylation tool and especially, to explore new means of epigenetic modulation, and its possible applications in editing hypermethylated genes for clinical purposes
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