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The understanding of epigenetic mechanisms in disease and development has surged in recent years with the introduction of better-optimized sample preparation methodologies and novel, high throughput sequencing workflows. Despite improvements in technologies that map epigenetic marks and interactions, few tools exist that facilitate functional studies of epigenetic marks in specific sequence contexts.
The relatively young field of genome editing has already led to many interesting findings. In most genome editing approaches, native chromosomes are manipulated to introduce new genetic sequence in a site-specific manner. In general this is accomplished with hybrid complex with two domains:
Researchers are deploying a number of approaches in labs today, but the most success has been with three types of methodologies:
Zinc finger proteins
Zinc finger proteins have been used extensively over the years. Various research teams have customized these proteins over the years leading to a fairly complete set of zinc finger domains available today that have binding specificities for nearly every nucleotide triplet (Pabo et al, Annu Rev Biochem, 2001). This broad availability has made zinc fingers a useful and flexible tool in genome editing.
Despite being one of the first tools to gain broader uptake in genome editing, zinc fingers still haven’t been widely adopted. This could be linked to the fact that labs often need to create custom fusions and screen the domains for efficacy or purchase a commercial solution.
Transcription activator-like effector nucleases (TALEs)
TALEs are a more recent addition to genome editing toolset. First reported in 2007,TALEs are natural DNA binding proteins found in plant pathogens. Much like zinc fingers, TALEs can be engineered to recognize specific DNA sequence motifs and they are modular, making them useful tools to target almost any motif in the genome.
TALEs and zinc fingers differ, however, in the way they recognize their binding sequences. Zinc finger domains recognize a triplet, whereas the active domains in TALEs recognize a single nucleotide, making it necessary to use a many TALEs to deliver the sequence specificity of fewer zinc fingers. That said, sometimes the triplet recognition of zinc fingers can make them less flexible for custom designs.
Although, TALEs have been used and studied for much less time that zinc fingers, early progress suggests that they may be at very least another useful tool in genome editing, if not a potential replacement for zinc fingers in some applications.
Clustered regularly interspaced short palindromic repeats
A new tool has recently captured the genome editing community’s attention. Clustered regularly interspaced short palindromic repeats CRISPR-Cas modules provide archaea and bacteria with adaptive immunity against invading pathogens. Although researchers have known of these repeats for some time and had even suggested functional importance over a decade ago (Mojica et al, Mol Microbiol. 2000), only recently has their utility in genome editing been reported.
Unlike zinc fingers and TALEs, the CRISPR-Cas system binds target DNA via small RNA guides instead of DNA binding proteins that introduces:
Collectively these benefits make the CRISPR-Cas system a viable contender for high throughput scenarios as well.
Understandably, the excitement in the genome editing field continues to grow with every advance and new tool that becomes available. Only recently have researchers begun pushing genome editing approaches into more advanced applications like epigenetic editing.
If genome editing is in its early years, epigenetic applications of genome editing tools could be considered embryonic, but that hasn’t stopped tech savvy researchers from pushing the limits. After all, epigenetic editing could address a bigger technology bottleneck in epigenetics than high throughput sequencing; that is if it provides a robust way to better assess the role of specific epigenetics marks throughout the epigenome; a technical hurdle that is all but impossible today.
Already, several viable routes of applying genome editing tools to epigenetics and ncRNA targets have emerged including:
Targeted DNA methylation editing with Zinc Fingers
The ability to direct DNA methylation site-specifically would provide researchers a valuable tool for interrogating the function of the mark in different conditions.
Over the past few years, researchers have had success using zinc finger proteins fused to various DNA methyltransferases in experiments that attempted to direct DNA methylation. These have been accomplished in a number of proof of principle contexts including oligonucleotide and reporter systems (de Groote et al, Nucleic Acids Research, 2012), but only recently have zinc fingers been used to direct DNA methylation in a more natural chromatin context.
In 2012 Rivenbark and colleagues used a zinc finger protein tethered to DNA methyltransferase 3a (DNMT3a) to target the tumor suppressor gene, Maspin and the oncogene SOX2. The research team was able to direct DNA methylation to the promoters of these genes that enabled targeted and specific repression (Rivenbark et al, Epigenetics, 2012).
Targeted DNA demethylation with Zinc Fingers
DNA methylation is a two way street in disease with many genes in displaying hypomethylation and hypermethylation. The recent discovery and subsequent studies of 5-hydroxylmethylcytosine (5-hmC), suggest that this novel mark is an intermediate in a broader demethylation pathway and that the Ten-Eleven Translocation (TET1, -2 or -3) family of 5-methylcytosine dioxygenases are key to the first step of this pathway; converting 5-mC to 5-hmC (Tahiliani et al, Science 2009).
Capitalizing on this, a team of researchers put the TET2 enzyme to work by fusing it to two zinc finger proteins targeting the promoters of InterCellular Adhesion Molecule-1 (ICAM-1) or Epithelial Cell Adhesion Molecule (EpCAM), two epigenetically silenced genes. In this study, the team was able to successfully demethylate several hypermethylated CpGs near the binding site in ICAM-1, but interestingly only one CpG in the EpCam promoter, an effect that the authors suggest may be related to a higher degree of hypermethylation in the EpCam promoter vs. the ICAM-1 promoter (Chen et al, Nucleic Acid Research, 2013).
Although, the targeted demethylation in this study wasn’t necessarily substantial, it did represent a major step forward in targeting demethylating enzymes to regions of interest in the genome.
TALEs of Epigenetic inhibition and ncRNA disruption
Unlike zinc fingers, TALEs have been utilized less to target epigenetic marks. This may be related to the fact that the technology was only recently introduced, but one study suggests that TALEs may run into issues, particularly with accessing epigenetically silenced regions.
In an interesting study of the oct4 pluripotency gene, Bultmann and colleagues, produced five TALEs targeting the gene. Although the TALEs exhibited strong similar binding affinities to the gene, they varied considerably in their ability to activate the silenced gene. Further investigation using a combination of histone deacetylase and DNA methylation transferase inhibitors indicated that this effect was likely due to local chromatin structure inhibiting the TALE mediated gene activation and that chemical manipulation might need to be added into the equation to use TALEs in epigenetically silenced targets (Bultmann et al, Nucleic Acids Research, 2012).
Although TALEs might run into a few issues with epigenetically silenced genes, they have been shown to be effective for studying non-coding RNA (ncRNA) function. A team of researchers led by Dr. Yun Liu recently used TALEs to create large genomic deletions for ncRNAs, including lncRNAs and miRNAs in zebrafish model system. These knockouts were inheritable and provided a useful approach for systematically studying function in ncRNAs (Liu et al, PLos One, 2013).
Epigenetics applications of genome editing are poised to transform the field. Zinc fingers and TALEs are already migrating through the proof of principle stage onward to more realistic and useful settings. CRISPR-Cas systems have already proven effective in a number of genetic applications in model organisms (Hwang et al, PLoS One, 2013, Jao et al, PNAS, 2013), but will CRISPR rise to the challenges involved in epigenetic editing, particularly in the context of heterochromatin? That’s a tough question to answer today, but one that is certainly being tackled in labs around the world this moment.