The definition of “epigenetics” is heavily debated116,117. Here, we use the word “epigenetic” to imply the molecular mechanism of heritable gene expression changes that cannot be attributed to changes in DNA sequence information. Unlike epigenetics, which implies the mechanism, the epigenome describes all post-translational modifications and other chromatin features associated with regulatory elements in the genome. Recent large-scale epigenomic efforts such as the Encyclopedia of DNA elements (ENCODE) and Roadmap Epigenome Mapping Consortium (REMC) efforts have mapped chromatin modifications both on DNA and histone proteins across the genome in various cell lines as well as primary cell types and tissues118,119. Although these epigenomic maps revealed unprecedented insight into cell-type-specific gene regulation and genome organization, the functional roles of various epigenomic features, such as histone modifications and DNA methylation, remain to be fully understood. To this end, locus-specific epigenome mapping tools and technologies are expected to greatly empower researchers to elucidate functional roles of chromatin modifications. Such tools will enable investigating some of the long-standing questions of chromatin biology such as the causal relationship between the presence of an epigenetic mark and gene expression. Furthermore, the ability the alter locus-specific epigenetic marks may enable us to identify the temporal kinetics of an epigenetic mark and its physical role on the functional epigenetic memory and gene expression. Therefore, soon after the CRISPR-Cas9 system was harnessed as an efficient gene-editing technology, researchers used the programmable capacity of dCas9 to recruit various epigenetic writers and erasers to a specific locus.

There are multiple layers of epigenetic regulatory mechanisms operating in the genome. Among the well-described ones are DNA methylation, histone posttranslational modifications, and non-coding RNAs (short and long). Among these, DNA methylation has the longest history, as researchers noticed and started to study its role in gene expression and development in the early 1970s120,121. DNA methylation is one of the most widely studied epigenetic mechanisms of gene regulation. Notably, in plants and other organisms, DNA methylation is found in three different sequence contexts: CG (or CpG), CHG, or CHH (H is A, T, or C), whereas in mammalian systems, the majority of DNA methylation happens at the fifth carbon of Cytosine residues (5-methylcytosine) of CpG dinucleotides122. DNMT3A and DNMT3B are the two DNA methyltransferase enzymes that catalyze de novo DNA methylation123. 5-Cytosine DNA methylation at promoter or distal regulatory elements is generally associated with transcriptional repression. Aberrant DNA methylation has been implicated in a number of pathological diseases including cancer. Therefore, there is strong unmet therapeutic need to manipulate aberrant disease-associated epigenomic features. In line with this, some of the small molecule epigenetic inhibitors that globally target DNA methylation such as 5-azacytidine are FDA approved124. Although such small molecules are already in clinical use, they target the entire genome and thus alter the chromatin state of loci where the epigenetic state is normal. Therefore, developing locus-specific epigenetic editing tools that specifically target aberrantly regulated loci has great therapeutic potential. To achieve this proof of principal, researchers utilized the dCas9 system to both deposit DNA methylation marks as well as remove the endogenous DNA methylation from the target site. To deposit DNA methylation at a specifically targeted locus, researchers fused dCas9 to the catalytic domain of eukaryotic DNA methyl transferase (DNMT3A)125,126,127,128,129,130,131,132 or prokaryotic DNA methyltransferase (MQ3)129. In both strategies, substantial deposition of DNA methylation and altered gene expression were observed at the target site. Importantly, targeted recruitment of additional components of repressive epigenetic machinery such as KRAB-ZNF, DNMT3L and polycomb complexes further enhanced the robustness of DNA methylation and long-term sustained gene repression126,133. These early proof of principle studies have reported highly specific deposition of DNA methylation at the target loci and local effects on gene expression. Interestingly, by using a DNA methyltransferase-deficient embryonic stem cell model, a recent study reported that dCas9-fused DNA methyl transferase has global off-target effects by leaving methylation footprints that are independent of sgRNA and methods of delivery134. Notably, despite the global increase in DNA methylation, which was attributed to abundant free nuclear dCas9 fused methyl transferase; limited impact on gene expression was observed134. It remains to be seen whether reducing the total free dCas9-fused methyl transferase will be as efficient and whether this pervasive global off-target effect is also a general characteristic of other epigenetic effectors. In addition to targeted DNA methylation, active removal of local methylation marks from endogenous loci is another strategy to manipulate gene expression through DNA methylation. Endogenous DNA demethylation is carried out by ten-eleven translocation (TET) proteins: TET1, TET2, and TET3. The proteins play a critical role in dynamic epigenetic regulation that mediates cell type-specific gene expression programs and lineage specification during development135. Therefore, a number of research groups aimed to achieve locus-specific DNA demethylation by using guidable dCas9 as a platform to recruit the catalytic domains of TET proteins125,131,132,136. Notably, the dCas9-TET1 fusion complex resulted in DNA demethylation in up to 90% of local CpG dinucleotides and a substantial increase in mRNA expression at the target sites125,131,132. Although robust locus-specific DNA demethylation and altered gene expression on target sites were reported, it remains to be seen whether dCas9-TET fusions may leave a global demethylation footprint akin to the methylation footprint of dCas9-fused methyltransferase.

In addition to DNA methylation, epigenetic information is stored in histone proteins, which DNA is wrapped around to form the chromatin fiber. Posttranslational modifications on histone tails constitute major epigenomic features that reveal key insights about regulatory activity of genomic elements. For example, active distal regulatory elements in the genome are marked with mono- and di-methylation at the lysine four position of Histone H3 (H3K4me1/2) and acetylation at the Lysine 27 acetylation position (H3K27ac), whereas active or poised bivalent promoters are marked with tri-methylation of Lysine four (H3K4me3)137. Chromatin modifications are dynamically regulated by various epigenetic writers, readers, and erasers138. Therefore, researchers are exploiting the versatile dCas9 platform to recruit various histone modifiers to a specific locus to better study the downstream effects of histone modifications. Research in this area has been focused on locally depositing histone methylation or acetylation as well as removing such marks. It should be noted that, unlike histone methylation, which could be associated with active and repressive chromatin features depending on the site of methylation, histone acetylation is observed at active gene promoters and enhancers.

In one study, researchers targeted a dCas9-LSD1 fusion complex to manipulate the regulatory activity of distal enhancer regions139. LSD1 is a histone demethylase that removes H3K4me2 mark140. In line with previous TALE-based LSD1 fusion studies141, dCas9-mediated locus-specific recruitment of LSD1 resulted in substantial local reduction in the active enhancer markers H3K4me2 and H3K27ac139 and altered expression of target genes. Active enhancer elements are marked by both H3K27ac as well as H3K4me1/2. Therefore, an alternative approach to epigenetically manipulating enhancer function is to locally deposit H3K27ac marks. Therefore, in contrast to the local reduction of enhancer marks by dCas9-LSD1, recruitment of histone acetyl transferase P300 through dCas9 fusion (dCas9-P300) resulted in a significant increase in local H3K27ac levels at enhancer elements142. Importantly, unlike other dCas9-fused transactivators, which can result in induction of gene expression primarily from promoter regions, targeting dCas9-P300 allows significant gene expression induction from both promoter and enhancer regions142. Researchers have also exploited other epigenetic modifiers to manipulate additional epigenetic marks. Among these, dCas9 fusion to the PRDM9 methyltransferase fusion complex has been utilized to manipulate local H3K4me3 marks143. Notably, local induction of H3K4me3, which is a marker of active promoters, was observed to be sufficient to allow re-expression of silenced target genes in various cell types143. Histone de-acetylation has been another strategy to locally manipulate chromatin structure and function. To this end, dCas9 fusion to histone deacetylases (HDAC), specifically full-length HDAC3, has been shown to effectively reduce the H3k27ac at the target loci and reduce the gene expression of the target loci144.

These aforementioned locus-specific epigenetic manipulation strategies are based on overexpression of a dCas9-fused epigenetic modifier complex. Such tools have been shown to specifically manipulate the expression of the target loci. However, whether overexpression of the fusion epigenetic complexes may leave a low level but global epigenetic footprint in the genome, as noted for the dCas9–DNMT3A fusion complex134, is yet to be determined. Therefore, novel strategies that enable local recruitment of endogenous epigenetic machineries may provide a higher precision in epigenetic editing. To this end, novel approaches such as Fkbp/Frb-based inducible recruitment for epigenome editing by Cas9 (FIRE–Cas9)145 may provide higher specificity in epigenetic editing by recruiting endogenous chromatin regulators.

Identifying the causal link between epigenetic marks and gene expression remains a central goal of chromatin biology. Thus, these aforementioned studies using dCas9 as a guidable platform to edit locus-specific epigenetic information will be an indispensable tool to achieve this. Now that the tools that enable us to alter the epigenome are in place, the next phase is to utilize them to better characterize regulatory elements and cellular states. To this end, researchers have already applied dCas9-based epigenome-editing tools for a number of exciting purposes including high-throughput screenings to characterize functional distal enhancers146, targeted reprogramming of lineage specification147,148, generation of induced pluripotent stem cells149, and reversal of HIV latency150. One of the remaining challenges is to elucidate the causal relationship between the presence of an epigenetic mark and its regulatory impact. Since the dCas9-fused epigenetic modifier remains associated with the target site, it is unclear whether the regulatory activity is due to the induced epigenetic mark or the complex. To this end, recent efforts using rapid and reversible epigenome-editing approaches are highly notable145. Future studies that enable rapid degradation of the targeting complex at the target site, such as with auxin-inducible degron technology151, should allow us to further characterize the functional consequences of epigenetic marks and investigate the associated temporal epigenetic memory for each mark.