Comparing genome-wide
epigenetic maps with
gene expression has allowed researchers to assign either activating or repressing roles to specific modifications. The importance of DNA sequence in regulating the epigenome has been demonstrated by using DNA motifs to predict epigenomic modification. Further insights into mechanisms behind
epigenetics have come from
in vitro biochemical and structural analyses. Using
model organisms, researchers have been able to describe the role of many
chromatin factors through
knockout studies. However knocking out an entire chromatin modifier has massive effects on the entire genome, which may not be an accurate representation of its function in a specific context. As one example of this,
DNA methylation occurs at
repeat regions,
promoters,
enhancers, and
gene bodies. Although
DNA methylation at gene promoters typically correlates with gene repression, methylation at gene bodies is correlated with gene activation, and DNA methylation may also play a role in gene splicing. The ability to directly target and edit individual methylation sites is critical to determining the exact function of DNA methylation at a specific site. Epigenome editing is a powerful tool that allows this type of analysis. For site-specific DNA methylation editing as well as for histone editing, genome editing systems have been adapted into epigene editing systems. In short, genome homing proteins with engineered or naturally occurring nuclease functions for gene editing, can be mutated and adapted into purely delivery systems. An epigenetic modifying
enzyme or domain can be fused to the homing protein and local epigenetic modifications can be altered upon protein recruitment. Exceptionally for
DNA methylation, the homing domain itself can be enough to interfere with normal epigenetic processes to lead to targeted epigenetic editing.
Targeting proteins TALE The
Transcription Activator-Like Effector (TALE) protein recognizes specific DNA sequences based on the composition of its
DNA binding domain. This allows the researcher to construct different TALE proteins to recognize a target DNA sequence by editing the TALE's primary
protein structure. The binding specificity of this protein is then typically confirmed using
Chromatin Immunoprecipitation (ChIP) and
Sanger sequencing of the resulting DNA fragment. This confirmation is still required on all TALE sequence recognition research. When used for epigenome editing, these DNA binding proteins are attached to an effector protein. Effector proteins that have been used for this purpose include
Ten-eleven translocation methylcytosine dioxygenase 1 (TET1), Kungulovski and Jeltsch successfully used ZFP-guided deposition of DNA methylation gene to cause
gene silencing but the DNA methylation and silencing were lost when the trigger signal stopped. The authors suggest for stable epigenetic changes, there must be either multiple depositions of DNA methylation of related epigenetic marks, or long-lasting trigger stimuli. ZFP epigenetic editing has shown potential to treat various neurodegenerative diseases.
CRISPR-Cas The
Clustered Regulatory Interspaced Short Palindromic Repeat (CRISPR)-Cas system functions as a DNA site-specific nuclease. In the well-studied type II CRISPR system, the Cas9 nuclease associates with a chimera composed of tracrRNA and crRNA. This chimera is frequently referred to as a guide RNA (gRNA). When the Cas9 protein associates with a DNA region-specific gRNA, the Cas9 cleaves DNA at targeted DNA loci. However, when the D10A and H840A
point mutations are introduced, a catalytically-dead Cas9 (dCas9) is generated that can bind DNA but will not cleave. The dCas9 system has been utilized for targeted epigenetic reprogramming in order to introduce site-specific DNA methylation. By fusing the
DNMT3a catalytic domain with the dCas9 protein, dCas9-DNMT3a is capable of achieving targeted DNA methylation of a targeted region as specified by the present guide RNA. Similarly, dCas9 has been fused with the catalytic core of the human acetyltransferase
p300. dCas9-p300 successfully catalyzes targeted acetylation of
histone H3 lysine 27. Alternatively, the dCas9 protein alone is sufficient to physically interfere with normal processes which maintain
DNA methylation at the site to which it is targeted in dividing cells; this results in targeted DNA demethylation. 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.
CRISPRoff is a dead Cas9
fusion protein that can be used to heritably silence the gene expression of "most genes" and allows for reversible modifications.
Commonly used effector proteins TET1 induces demethylation of cytosine at
CpG sites. This protein has been used to activate genes that are repressed by CpG methylation and to determine the role of individual CpG methylation sites. The interaction allows the chromatin modifier to act on the desired location. This means that the modification can be performed in an inducible and reversible manner, which reduces long-term secondary effects that would be caused by constitutive epigenetic modification. == Applications ==