Engineering CpG island DNA methylation in pluripotent cells through synthetic CpG-free ssDNA insertion

Transgenerational Epigenetic DNA Methylation Editing and Human Disease

“…Overall, programmable transgenerational DNAme editing is possible, but whether these approaches can be generalized genome-wide remains to be determined. We have similarly observed CG-free insertion by synthetic CpG-free ssDNA insertion in both human and mouse embryonic stem cells to induce CGI-wide, stable, and globally specific DNAme [8]. Functionally retained through in vitro differentiation, engineered DNAme is retained post-CG-free DNA removal and through multilineage differentiation.…”

“…Functionally retained through in vitro differentiation, engineered DNAme is retained post-CG-free DNA removal and through multilineage differentiation. Furthermore, designer MLH1 promoter DNAme was observed to skew thymic epithelial cell differentiation and sensitize multiple lineages to cisplatin [8]. By transcription factor binding enrichment analysis, we identified KLF6 as having a putative role in regulating CIMR responses.…”

“…With regards to developmental CIMR timing, blastocysts did not exhibit targeted DNAme of the Ldlr CGI, but epiblasts did; thus, acquired DNAme at these interrupted CGIs occurs after implantation, upon re-establishment of global DNAme. In hESCs, we have observed CIMR DNAme acquisition to be restricted to the primed state of pluripotency and for reacquisition to occur when hESC transition from naïve to primed states using defined cultures ( [8,15] unpublished observations). Collectively, this suggests that CG-free-DNA insertion into CGI or CGdense areas of the genome, in germ cells or zygotes, will trigger locus-or region-specific DNAme as embryos transition through primed stages of development.…”

“…Major epigenetic factors include covalent DNA modifications, such as DNA methylation (DNAme) or quadruplex structures, a multitude of histone modifications and chromatin remodelers, which facilitate DNA accessibility, as well as short-and long-noncoding RNAs that mediate DNA-protein interactions [1][2][3][4][5]. There are some cells which maintain epigenome plasticity over their lifetime (e.g., multipotent stem cells), while others exhibit terminal post-mitotic configurations (e.g., cardiomyocytes) [6][7][8][9][10][11][12]. Primordial germ cells (PGCs) and cells of the pre-implantation blastocyst stage of development will uniquely undergo a remarkable form of epigenetic remodeling, in which genome presentation is reset, and the genome evolves between parent and child [11][12][13][14][15].…”

“…Reminiscent of repetitive element silencing, Takahashi et al, recently identified that CG-free DNA insertion into CGIs triggered CGI-specific and CGI-wide de novo DNAme in both human PSCs and mouse PSCs [8,58] (Figure 6). Remarkably, mouse blastocyst/mPSC chimera offspring retained DNAme edits, which, despite the subsequent removal of the inserted DNA in mPSCs, was transgenerationally inherited [58].…”