Regulation of gene expression by altered promoter methylation using a CRISPR/Cas9-mediated epigenetic editing system

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.

Therapeutic applications of CRISPR/Cas9 in breast cancer and delivery potential of gold nanomaterials

“…DNA methylation commonly occurs on the C of CpG repeats in promoter regions, called CpG islands, preventing TF binding and thus inhibiting gene expression. 73 The process is carried out by DNMT1, DNMT3A, and DNMT3B enzymes, which convert C to 5 0 -methylcytosine (5 0 -mC) and ensure methylation is maintained during cell division. 74 In addition to TSG hypermethylation, hypomethylation at the promotor level, facilitating expression of genes promoting tumour growth; and at the genome level, potentially destabilising chromosomes, have been observed in BCs.…”

Various Aspects of a Gene Editing System—CRISPR–Cas9

“…In a different study, scientists demonstrated the utility of the CRISPR/Cas9 system for modulating methylation at specific CpG sites and inducing gene expression. They targeted murine Oct4 gene, which is transcriptionally blocked, due to hypermethylation in the promoter region in NIH3T3 cell line [ 51 ]. Oct4 is only expressed in stem cells and is responsible for self-renewal and the maintenance of the pluripotent state of stem cells.…”

Combining Multiple Approaches and Models to Dissect the Genetic Architecture of Resistance to Infections in Fish

“…Genome-wide KO in vitro screen is also possible in well-established mammalian–lentivirus systems ( Shalem et al, 2014 , 2015 ) and may be applicable in fish cells in the near future ( Gratacap et al, 2020 ). Genome editing can also target non-coding sequences and contribute to validation of mutation in, for instance, regulatory sequences ( Banerjee and Sherwood, 2017 ; Kang et al, 2019 ).…”