**3.1. Transcriptional activation**

Transcriptional activator effector domains were first used to upregulate levels of gene expression by fusing the herpes simplex virus protein VP16—involved in recruitment of chromatin remodeling factors that increase chromatin accessibility—and its VP64 tetrameric form to zinc finger proteins [41, 42]. For instance, an early study demonstrated that zinc finger proteins targeted to 18 base-pair sequences of the 5′ untranslated region of the proto-oncogene *erbB-2* could upregulate transcription in an endogenous context [41]. Upregulation was also shown to occur even under control of an exogenous chemical inducer molecule [41]. Similarly, zinc finger fusions to the VP64 activation domain have been used to reactivate dormant genes. In one study, a dormant mammary serine protease inhibitor (*maspin*) tumor suppressor gene was reactivated by targeting the *maspin* promoter region, which is often epigenetically silenced in aggressive epithelial tumors [43]. Other transcriptional activator domains, such as the p65 subunit of the NF-κB complex, have also been coupled to zinc finger proteins for targeted transcriptional activation [44].

The proof-of-concept studies using zinc fingers fused to transcriptional activation domains to modulate gene expression patterns served as blueprints for the subsequent use of activator domain fusions to TALEs and CRISPR–dCas9 systems. TALE activators targeted to promoter sites of the endogenous human *VEGF-A*, *NTF3,* and the microRNA *miR-302/367* cluster have been reported to induce increased target gene expression by fivefold or more [28]. Endogenous levels of gene expression of select human pluripotency factors have also been shown to increase by twofold to fivefold using TALE fusions to VP64 [45].

Furthermore, although single TALE fusions to transcriptional activators have demonstrated the ability to upregulate gene expression, combinations of TALE-VP64 fusions targeting the promoter regions of genes implicated in inflammation, immunomodulation, and cancer pathways have established the occurrence of synergistic activation effects at target sites, which presents opportunities to develop tunable transcriptional networks [46]. Interestingly, it appears that different types of transcriptional activators induce varying levels of gene expression. In one study, the mean-fold activation induced with TALE-p65 activators was lower than that with TALE-VP64 activators [28]. The combination of VP64 and p65 activators exhibits synergistic effects on endogenous gene expression, which can provide a platform not only to design tools for targeted gene expression, but also to finetune the fold activation induced by different combinations of activator domains fused to DNA-binding proteins [28].

Recently, researchers have built upon work on zinc finger and TALE fusions to develop technologies for targeted gene expression utilizing the CRISPR–dCas9 system. RNA-guided transcriptional upregulation using CRISPR–dCas9-VP64 and CRISPR–dCas9-p65 fusions has been reported by several research teams in the last 5 years [47–50]. Introduction of individual sgRNAs for dCas9 targeting can induce transcriptional activity at desired loci, but the use of multiple sgRNAs to target a single locus can act synergistically to induce multifold gene expression [48–50]. The CRISPR–dCas9 system offers several advantages over zinc fingers and TALEs for targeted gene activation. Because CRISPR–dCas9 is an RNA-guided, DNAtargeting system, it does not require complex protein engineering and could open additional research paths for spatiotemporal control of gene expression alongside complex chemical and optogenetic inducible systems. However, some studies have reported that dCas9-based activation tools induce weaker levels of transcription compared to TALE-based activators [50, 51].

In addition to the use of transcriptional activators for targeted gene expression, research teams have demonstrated the efficacy of using epigenetic effectors fused to DNA targeting proteins to manipulate epigenetic landscapes. In 2015, a study reported the use of CRISPR–dCas9, TALE, and zinc finger fusions to the p300 histone acetyltransferase (HAT) catalytic domain, which deposits H3K27 and other acetylation marks on histone tails, to trigger transactivation of genes [39]. Notably, the study showed that CRISPR–dCas9-p300 Core fusions led to more efficient and robust activation from proximal and distal enhancer regions compared to synthetic transcription factors engineered to carry activation domains that target promoters. Other epigenetic effectors that catalyze covalent modifications on DNA have also been fused to DNA-binding proteins to induce gene expression. For example, ten-eleven translocation (TET) demethylase enzymes that catalyze the sequential oxidation of 5-methyldeoxycytosine (m<sup>5</sup> dC) to form 5-hydroxymethylcytosine (hm<sup>5</sup> C), 5-formylcytosine (f<sup>5</sup> C), and 5-carboxylcytosine (ca<sup>5</sup> C) have been used with zinc fingers [52–54], TALEs [55], and CRISPR–dCas9 [56, 57] to direct promoter-specific DNA demethylation and, thus, upregulate gene expression.

As mentioned earlier, CRISPR–dCas9 systems represent promising tools for the next generation of complex inducible systems. More recently, a novel system comprising CRISPR–dCas9 fused to a tripartite activator featuring the constituent activation domains VP64, p65, and Rta (VPR) was shown to activate endogenous coding and noncoding regions while targeting several genes simultaneously [58]. Another platform built with CRISPR–dCas9 and a protein scaffold—namely, a repeating peptide array called the SUperNova tagging system (SunTag)—capable of recruiting multiple copies of an antibody fusion protein showed robust levels of endogenous gene activation at target sites [59]. Lastly, it has been demonstrated that CRISPR–Cas sgRNAs can be engineered to carry protein-binding cassettes, artificial aptamers, and other ncRNAs directly into the sgRNA stem-loop structures, thereby creating modular CRISPR–dCas9 complexes that enable locus targeting, multiplexing, and highly inducible regulatory action to upregulate patterns of gene expression [60–62].

### **3.2. Transcriptional repression**

**3. Epigenome editing to regulate gene expression**

**3.1. Transcriptional activation**

152 Chromatin and Epigenetics

transcriptional activation [44].

DNA-binding proteins [28].

Manipulation of endogenous gene expression at target loci has thus far been achieved by coupling DNA-binding proteins with (1) transcriptional activators and repressors, which modulate gene expression by acting as site-specific ATFs, or (2) epigenetic effectors, which catalyze the deposition or removal of specific epigenetic modifications at target loci. In addition, the CRISPR–dCas9 system has recently enabled engineering of modular sgRNA molecules that carry RNA aptamers as scaffolding platforms to recruit molecules for site-specific regulation.

Transcriptional activator effector domains were first used to upregulate levels of gene expression by fusing the herpes simplex virus protein VP16—involved in recruitment of chromatin remodeling factors that increase chromatin accessibility—and its VP64 tetrameric form to zinc finger proteins [41, 42]. For instance, an early study demonstrated that zinc finger proteins targeted to 18 base-pair sequences of the 5′ untranslated region of the proto-oncogene *erbB-2* could upregulate transcription in an endogenous context [41]. Upregulation was also shown to occur even under control of an exogenous chemical inducer molecule [41]. Similarly, zinc finger fusions to the VP64 activation domain have been used to reactivate dormant genes. In one study, a dormant mammary serine protease inhibitor (*maspin*) tumor suppressor gene was reactivated by targeting the *maspin* promoter region, which is often epigenetically silenced in aggressive epithelial tumors [43]. Other transcriptional activator domains, such as the p65 subunit of the NF-κB complex, have also been coupled to zinc finger proteins for targeted

The proof-of-concept studies using zinc fingers fused to transcriptional activation domains to modulate gene expression patterns served as blueprints for the subsequent use of activator domain fusions to TALEs and CRISPR–dCas9 systems. TALE activators targeted to promoter sites of the endogenous human *VEGF-A*, *NTF3,* and the microRNA *miR-302/367* cluster have been reported to induce increased target gene expression by fivefold or more [28]. Endogenous levels of gene expression of select human pluripotency factors have also

Furthermore, although single TALE fusions to transcriptional activators have demonstrated the ability to upregulate gene expression, combinations of TALE-VP64 fusions targeting the promoter regions of genes implicated in inflammation, immunomodulation, and cancer pathways have established the occurrence of synergistic activation effects at target sites, which presents opportunities to develop tunable transcriptional networks [46]. Interestingly, it appears that different types of transcriptional activators induce varying levels of gene expression. In one study, the mean-fold activation induced with TALE-p65 activators was lower than that with TALE-VP64 activators [28]. The combination of VP64 and p65 activators exhibits synergistic effects on endogenous gene expression, which can provide a platform not only to design tools for targeted gene expression, but also to finetune the fold activation induced by different combinations of activator domains fused to

been shown to increase by twofold to fivefold using TALE fusions to VP64 [45].

Silencing endogenous gene expression at target loci has been achieved in similar ways to gene activation by fusing repressor domains or epigenetic effectors to DNA-binding proteins. The domain most commonly used to trigger gene silencing is the Krüppel-associated box (KRAB). KRAB repression is mediated by the recruitment of complexes that elicit formation of heterochromatin via interactions between the KRAB-associated protein 1 (KAP1) corepressor and other factors that catalyze histone methylation and deacetylation [17, 63]. KRAB fusions to zinc finger proteins [41, 64], TALEs [65], and CRISPR–dCas9 [40, 47] have demonstrated robust inactivation from promoter as well as proximal and distal regulatory elements.

remains a nascent field that must overcome many technological challenges related to editing

Epigenome Editing

155

http://dx.doi.org/10.5772/intechopen.79564

A major current limitation concerns the spatio-temporal specificity of epigenome editing, which may involve multiple factors including, but not limited to, non-specific deposition or removal of select epigenetic marks on target DNA loci or histones, mitigation of off-target DNA binding effects, and temporal expression of epigenetic effectors in different cell types and chromatin contexts [3]. For example, enzymes that catalyze the deposition of histone PTMs—e.g., p300's HAT domain, which catalyzes acetylation of multiple residues on all four core histones—have various degrees of histone substrate specificity. Thus, the inherent promiscuity of an enzyme may pose challenges to the study of direct functional roles for unique

Similarly, the expression levels and times at which an epigenetic effector is present in a cell type can exert effects on the efficiency of DNA binding specificity. Following target site saturation, off-target binding activity is likely to increase if the effector remains, or continues to be expressed, in the cell. This could partly explain the extensive global off-target effects reported with the use of dCas9–methyltransferase fusions in one study [73]. Given the lack of target site specificity previously reported for dCas9–sgRNA complexes [74], further research is needed to establish whether other dCas9–effector fusions exhibit similar off-target activities. The use of zinc finger and TALE fusions to epigenetic effectors offers an alternative to mitigate possible dCas9-related off-target effects. However, zinc finger and TALE epigenome editing biotechnologies are limited by the complex protein engineering required to mediate DNA

Progress in other areas associated with challenges in delivery, efficiency, and stability of epigenetic states has come at a rapid pace [75–77]. Chemical and optogenetic molecules have expanded the toolbox for reversible and inducible epigenome editing in endogenous contexts [3, 66, 78]. And emerging technologies for RNA editing [79] could soon provide new insights about the epigenetic roles of ncRNAs. As the field matures, epigenome editing will undoubtedly make significant contributions to the advancement of basic and applied

Recent technological breakthroughs in epigenome editing have expanded our understanding of the underlying mechanisms responsible for the regulation of chromatin structure and the spatiotemporal control of gene activation and repression. As detailed in this chapter, modern epigenome editing systems are revolutionizing the field of epigenetics. Programmable DNAbinding proteins fused to catalytically active or scaffolding epigenetic effectors represent invaluable tools to uncover the functional significance of site-specific epigenetic mechanisms in a myriad of contexts including development, cell differentiation, and disease. Undoubtedly, progress in this emerging field will offer great contributions to translational medicine and

efficiency, delivery, cytotoxicity, and specificity.

PTMs in specific contexts [3].

recognition.

research.

**4. Conclusion**

biotechnology in the near future.

Although the KRAB repressor is the most common domain used for recruitment of heterochromatin-forming factors, researchers have also reported efficient gene silencing with other repressor domains such as the mSin interaction domain (SID) [65] and Mxi1 [47]. One study comparing dCas9-KRAB and dCas9-Mxi1 showed that the Mxi1 fusion could induce more repression by nearly threefold levels [47]. A separate study found that TALE-SID repressors could lead to 26% more transcriptional repression than its TALE-KRAB counterparts [65]. Moreover, concatenating four SID (SID4X) domains—much like combining VP16 units into VP64 activator proteins—and fusing them to DNA-binding proteins can induce greater repression than single SID domain fusions [66].

In contrast to transcriptional repressors, which aptly recruit heterochromatin-forming complexes to desired loci, epigenetic effectors can directly catalyze specific modifications on histones and DNA that trigger targeted epigenetic repression. Several studies have thus far tested and confirmed robust repression efficiency rates of histone methyltransferases [17], histone demethylases [29, 67], and DNA methyltransferases [68–70] with zinc finger, TALE, and CRISPR–dCas9 proteins at target promoter and enhancer regions.

Notably, a study featuring a CRISPR–dCas9 fusion to the catalytic domain of DNMT3A, a de novo DNA methyltransferase, found that multiplexing with several sgRNAs exhibits synergistic effects on site-specific CpG methylation [70]. Thus, CRISPR–dCas9-DNMT3A multiplexing can lead to a greater increase of methylation levels at target regions. Despite the synergistic effects seen with DNMT3A repressor fusions, it appears that dCas9- DNMT3A epigenetic editing of target CpG methylation sites is not wholly stable and does not persist beyond the loss of expression of the editing construct [70]. To overcome this type of transient epigenetic editing, a study recently reported a system for inheritable silencing of endogenous genes by transiently expressing combinations of DNA-binding proteins fused to transcriptional repressors, including KRAB and DNMT3 domains, to prompt long-term memory of repressive epigenetic states [71]. Lastly, the modular nature of sgRNAs has enabled the creation of scaffolding systems to achieve multiplexing and multimerization of epigenetic effectors, which will be useful to study endogenous and synthetic gene repression [62, 72].

### **3.3. Technological limitations**

Epigenome editing directed by catalytically active or scaffolding epigenetic effectors fused to programmable DNA binding proteins has contributed important insights about the causal relationships between epigenetic states and gene regulation. However, despite the progress made in recent years to uncover the molecular basis of epigenetic processes, epigenome editing remains a nascent field that must overcome many technological challenges related to editing efficiency, delivery, cytotoxicity, and specificity.

A major current limitation concerns the spatio-temporal specificity of epigenome editing, which may involve multiple factors including, but not limited to, non-specific deposition or removal of select epigenetic marks on target DNA loci or histones, mitigation of off-target DNA binding effects, and temporal expression of epigenetic effectors in different cell types and chromatin contexts [3]. For example, enzymes that catalyze the deposition of histone PTMs—e.g., p300's HAT domain, which catalyzes acetylation of multiple residues on all four core histones—have various degrees of histone substrate specificity. Thus, the inherent promiscuity of an enzyme may pose challenges to the study of direct functional roles for unique PTMs in specific contexts [3].

Similarly, the expression levels and times at which an epigenetic effector is present in a cell type can exert effects on the efficiency of DNA binding specificity. Following target site saturation, off-target binding activity is likely to increase if the effector remains, or continues to be expressed, in the cell. This could partly explain the extensive global off-target effects reported with the use of dCas9–methyltransferase fusions in one study [73]. Given the lack of target site specificity previously reported for dCas9–sgRNA complexes [74], further research is needed to establish whether other dCas9–effector fusions exhibit similar off-target activities. The use of zinc finger and TALE fusions to epigenetic effectors offers an alternative to mitigate possible dCas9-related off-target effects. However, zinc finger and TALE epigenome editing biotechnologies are limited by the complex protein engineering required to mediate DNA recognition.

Progress in other areas associated with challenges in delivery, efficiency, and stability of epigenetic states has come at a rapid pace [75–77]. Chemical and optogenetic molecules have expanded the toolbox for reversible and inducible epigenome editing in endogenous contexts [3, 66, 78]. And emerging technologies for RNA editing [79] could soon provide new insights about the epigenetic roles of ncRNAs. As the field matures, epigenome editing will undoubtedly make significant contributions to the advancement of basic and applied research.
