Epigenome editing
By "editing” epigenomic features in this manner, researchers can determine the exact biological role of an epigenetic modification at the site in question.
Comparing genome-wide epigenetic maps with gene expression has allowed researchers to assign either activating or repressing roles to specific modifications.
In short, genome homing proteins with engineered or naturally occurring nuclease functions for gene editing, can be mutated and adapted into purely delivery systems.
The binding specificity of this protein is then typically confirmed using Chromatin Immunoprecipitation (ChIP) and Sanger sequencing of the resulting DNA fragment.
Chen et al. have successfully used a zinc finger DNA binding domain coupled with the TET1 protein to induce demethylation of several previously silenced genes.
[11] The Clustered Regulatory Interspaced Short Palindromic Repeat (CRISPR)-Cas system functions as a DNA site-specific nuclease.
[12] In the well-studied type II CRISPR system, the Cas9 nuclease associates with a chimera composed of tracrRNA and crRNA.
The primary benefit of this approach is that it is free of epigenetic-modifying enzymes, which may affect epigenetic marks over large distances and act independently throughout the genome despite being tethered to a targeted dCas9 protein, often leading to widespread off-target effects.
[3] A variant in CRISPR epigenome editing (called FIRE-Cas9) allows to reverse the changes made, in case something went wrong.
[5] Editing of gene enhancer regions in the genome through targeted epigenetic modification has been demonstrated by Mendenhall et al.
To study this, one research group used a TALE-TET1 fusion protein to demethylate a single CpG methylation site.
One research group employed an optogenetic two-hybrid system which integrated the sequence specific TALE DNA-binding domain with a light-sensitive cryptochrome 2 protein (CIB1).
[5] Once expressed in the cells, the system was able to inducibly edit histone modifications and determine their function in a specific context.
[22] While transgenerational and population level consequences are not fully understood, it may become a major tool for applied functional genomics and personalized medicine.
[8] In addition, DNA repair systems could reverse the alterations on the chromatin and prevent the desired changes from being made.
When these fusions are over-expressed in cells, these enzymes tend to modify large spans of the genome in what constitutes dramatic off-target activity.
