Targeted DNA Demethylation: Vectors, Effectors and Perspectives

Abstract

Aberrant DNA hypermethylation at regulatory cis-elements of particular genes is seen in a plethora of pathological conditions including cardiovascular, neurological, immunological, gastrointestinal and renal diseases, as well as in cancer, diabetes and others. Thus, approaches for experimental and therapeutic DNA demethylation have a great potential to demonstrate mechanistic importance, and even causality of epigenetic alterations, and may open novel avenues to epigenetic cures. However, existing methods based on DNA methyltransferase inhibitors that elicit genome-wide demethylation are not suitable for treatment of diseases with specific epimutations and provide a limited experimental value. Therefore, gene-specific epigenetic editing is a critical approach for epigenetic re-activation of silenced genes. Site-specific demethylation can be achieved by utilizing sequence-dependent DNA-binding molecules such as zinc finger protein array (ZFA), transcription activator-like effector (TALE) and clustered regularly interspaced short palindromic repeat-associated dead Cas9 (CRISPR/dCas9). Synthetic proteins, where these DNA-binding domains are fused with the DNA demethylases such as ten-eleven translocation (Tet) and thymine DNA glycosylase (TDG) enzymes, successfully induced or enhanced transcriptional responsiveness at targeted loci. However, a number of challenges, including the dependence on transgenesis for delivery of the fusion constructs, remain issues to be solved. In this review, we detail current and potential approaches to gene-specific DNA demethylation as a novel epigenetic editing-based therapeutic strategy.

Progress and Prospects of Gene Editing in Pluripotent Stem Cells

“…If both HNH and RuvC domains are inactivated, the enzymatically dead Cas9, dCas9, could serve as a scaffold for recruiting effectors to the desired site without making DNA breaks. Depending on the factors fused with, dCas9 can be used for activating (CRISPRa) or suppressing (CRISPRi) gene expression, epigenetic modification (e.g., DNA methylation or histone modifications), or molecular imaging [ 85 , 86 , 87 ]. Although epigenetic regulations play key roles in PSC functions, applications excellently reviewed elsewhere are omitted in the present review due to the space limitation and the absence of permanent alterations of DNA sequences induced by these variants.…”

Advantages and Challenges of CRISPR-Cas9 Applications in Animal Modeling: A Concise Review CRISPR-Cas9 Applications in Animal Modeling

“…Since each FokI nuclease makes a nick in one strand, for having double-strand breaks at a target site, two molecules of ZFNs are needed. Naturally, the cell tries to repair these breaks with a non-homologous end joining (NHEJ) pathway; however, in the presence of a DNA sequence complementary to the break sites, the homology-directed repair pathway (HDR) inserts a new sequence at the break sites (24)(25)(26).…”

“…Cas9 protein is a golden tool for genetic engineering and genetically modified organisms. Owing to its programmability, Cas9 has been proposed as a useful molecular tool in genomic editing in a variety of organisms and cells, including human cells, mice, zebrafish, Drosophila (20,21),Caenorhabditis elegans, rats (22), pigs (23), monkeys (24), and plant cells. Both in vitro and in vivo genome manipulation modes are easily possible using guide RNAs, artificially designed and synthesized gRNAs.…”

Smoking-Induced DNA Hydroxymethylation Signature Is Less Pronounced than True DNA Methylation: The Population-Based KORA Fit Cohort

“…During active demethylation process, the ten-eleven translocation (TET) enzymes play a crucial role by oxidizing 5mC into 5hmC, further converting 5hmC to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Subsequently, the thymine DNA glycosylase (TDG)-dependent base excision repair (BER) transforms 5fC and 5caC into an unmethylated cytosine [ 14 , 15 ]. Due to their low abundance in the genome, 5fC and 5caC demonstrate limited stability [ 16 ].…”