**2. Programmable epigenome editing systems**

Regulating endogenous levels of gene expression by targeting specific epigenetic modifications is a relatively nascent field. Basic scientific research in the last few decades has provided insights that facilitated the development of technologies aimed at interrogating epigenetic processes. At their core, epigenome editing tools are based on the concept of fusing programmable DNAbinding proteins that target specific genetic loci, with catalytically active or scaffolding effector domains that exert some influence on epigenetic processes. The three most important molecular tools that have been developed for targeted epigenome editing are zinc finger proteins, TALEs, and CRISPR–Cas systems. All have been repurposed into epigenome editing platforms designed to manipulate gene expression patterns in particular contexts. An overview of each system is provided below. However, it should be noted that other, less common, tools have also been developed to probe epigenetic mechanisms and modulate gene expression in a sequence-dependent manner including synthetic polyamides [8] and triple helix-forming oligonucleotides (TFOs) [9].

### **2.1. Zinc finger-based systems**

differentiation [1]. Waddington's "epigenetic landscapes" [2] laid a foundation for exploring phenotypic plasticity as a phenomenon that is not entirely dependent on genetic mechanisms, but one which can also be shaped by environmental cues that concomitantly orchestrate the

The scope of epigenetics as a scientific discipline, much like its definition, has expanded with the passage of time. Today, the field broadly refers to the study of fundamental processes related to mitotic and meiotic stable and heritable changes that emerge without alteration of DNA sequences [3–5]. Stable changes encompass durable alterations in gene expression patterns, which may be neither permanent nor heritable. In contrast, heritable changes in gene expression may constitute persistent alterations that are carried onto the progeny of cells or individual organisms [6].

In the last two decades, the emerging field of epigenetics has revealed crucial information about the regulation of chromatin states in the eukaryotic nucleus. Heterochromatic DNA is organized into compact, higher order, chromatin fibers. By contrast, euchromatic DNA comprises lightly packed chromatin that represents an active and accessible part of the genome. Both states feature an array of nucleosomes, the basic subunits of chromatin, consisting of approximately 145–147 base pairs of DNA wrapped around a core histone octamer [7]. Each histone octamer carries two copies of all of the core histone proteins—H2A, H2B, H3, H4 assembled into nucleosomes, which are subsequently stabilized into high-order chromatin

The organization and regulation of chromatin at the epigenetic level depend on dynamic and diverse combinations of covalent chemical modifications that occur on histone proteins known as histone post-translational modifications (PTMs)—and DNA bases, as well as the expression of regulatory noncoding RNA (ncRNA) molecules [3]. Together, these epigenetic signals and ncRNAs constitute a layer of information that controls the spatiotemporal regulation of gene expression patterns by remodeling the structure of chromatin and modulating its

The scientific community has made great strides in elucidating functional roles for epigenetic processes in recent years. Progress has come at the hand of biotechnologies aimed at manipulating endogenous, site-specific epigenetic targets. The technologies rely on the use of catalytically active or scaffolding epigenetic effectors fused to programmable DNA-binding proteins that target specific genetic loci. This chapter focuses on the three most important platforms for modern targeted epigenome editing: zinc finger proteins, transcription activator-like effectors (TALEs), and clustered, regularly interspaced, short palindromic repeats (CRISPR), and

Notwithstanding the progress made in recent years, much remains to be learned about epigenetic mechanisms in development, cellular programming, disease, and personalized medicine. Epigenome editing technologies are poised to make significant contributions to the field of epigenetics. This chapter provides an overview of each epigenome editing system, highlights their use for manipulating specific patterns of gene expression, and discusses the technological limitations that have thus far limited our ability to interrogate the mechanisms

process of cell differentiation from an initial totipotent state [3].

structures by the presence of the linker histone H1 and linker DNA [7].

CRISPR-associated sequences (Cas) (CRISPR–Cas) systems.

of epigenetic regulation in local and genome-wide contexts.

bio-physicochemical properties.

146 Chromatin and Epigenetics

Zinc finger proteins are among the most characterized systems used for the manipulation of targeted, sequence-specific nucleic acids. Their discovery arose from research of transcriptional mechanisms in *Xenopus laevis* oocytes dating back to over three decades ago. At the time, scientists realized that a unique set of proteins, containing repetitive zinc-binding domains of roughly 30 amino acid residues, was required for transcription factor-mediated gene regulation [10]. These finger-like peptide arrangements rely on interactions between conserved pairs of cysteine and histidine residues that are anchored by a centered and tetrahedrally coordinated zinc ion (**Figure 1**). From a structural standpoint, each zinc finger

**Figure 1. Crystal structure of two zinc finger proteins in complex with DNA**. (A) Recognition of target DNA (blue) by two distinct zinc finger proteins (orange and green). A tetrahedrally coordinated zinc ion (red) stabilizes each zinc finger motif. (B) Orthogonal view of zinc finger-mediated DNA recognition. [PDB 3DFX].

comprises an antiparallel β sheet and α helix, which are stabilized by the zinc ion and a set of hydrophobic residues [11]. The α helix of each zinc finger binds directly to the major groove of B-DNA. Residues from the NH<sup>2</sup> -terminal region of the helix mediate recognition of a three base-pair target site [11]. Thus, side chain residues in the α helix that can be engineered to recognize diverse nucleic acid base-pair triplets drive the double-stranded DNA sequence specificity of each zinc finger.

The molecular basis for DNA recognition of each TALE comes from a central tandem amino acid repeat domain of approximately 33–35 residues in length [23, 24]. This tandem repeat is flanked by an N-terminal region required for type III secretion and a C-terminal region involved in nuclear localization and transcriptional activation. Each repeat folds into two left-handed α helices linked by a short loop that contains two hypervariable residues, known as the repeat variable diresidues (RVDs). The RVDs occupy positions 12 and 13 of each repeat, where the 12th residue stabilizes the RVD loop by mediating contacts with the protein backbone, while the 13th residue interacts directly with a specific nucleotide nitrogenous base (**Figure 2**). Thus, the DNA specificity of TALEs is modular and encoded in the tandem repeat sequence [25, 26]. Unlike zinc fingers, which require triplet sequence recognition sites, TALEs are able to target a single nucleotide at a time through its RVDs. This characteristic makes TALEs simpler to engineer and has enabled the rational design of artificial TALEs for biotechnological applications [26]. Indeed, borrowing from its ZFN predecessors, TALE proteins fused to the *Fok*I nuclease domain gave rise to TALE nucleases (TALENs), which have been utilized for genome editing

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Similarly, fusions of TALEs to epigenetic effectors have been repurposed for epigenome editing to manipulate gene expression. For example, TALE fusions to activation domains have shown the ability to target specific genomic loci and induce robust transcriptional activation [28]. Inactivation of enhancer regulatory elements has also been demonstrated using TALE fusions to chromatin effectors targeting specific histone modifications [29], thereby establishing a platform to interrogate the function of specific regulatory elements in diverse chromatin landscapes.

**Figure 2. Structure of a TAL effector bound to its target DNA**. (A) The TALE tandem amino acid repeats (multi-colored helices) associate into a right-handed superhelix that wraps around the major groove of the target DNA (blue). (B)

Orthogonal view of target DNA recognition by a TALE [PDB 3UGM].

applications [27].

The intrinsic modularity of DNA recognition by zinc finger proteins led to the swift development of a range of biotechnological applications. For example, manipulation of gene expression via site-specific, DNA-binding zinc finger proteins was first established by targeting a 9 base-pair region of a *BCR*-*ABL* fusion oncogene [12]. Using a three-zinc finger peptide, scientists demonstrated that transcriptional repression of a leukemic oncogene in a chromosomal DNA context was possible [12].

Similarly, the development of zinc finger nucleases (ZFNs) marked an important milestone for modern genome editing biotechnologies. ZFNs are engineered chimeric fusions composed of a set of tandem zinc finger DNA-binding proteins and a nuclease domain—such as the catalytic domain of the restriction endonuclease *Fok*I—that cleaves DNA [13]. Synthetic zinc finger-*Fok*I fusions coupled the DNA sequence specificity of zinc finger proteins with the nonspecific cleavage activity of *Fok*I to trigger double-stranded breaks at desired genomic loci. Importantly, repurposing zinc finger proteins into ZFNs facilitated the path for epigenome editing biotechnologies aimed at controlling transcriptional activation and repression.

Zinc finger-related epigenome editing tools are based on the concept of fusing programmable, DNA binding, zinc finger proteins designed to target diverse sequences [14, 15] with catalytically active or scaffolding effector domains. The chimeric proteins are designed to alter gene expression patterns and act as artificial transcription factors (ATFs) [15]. Pioneer studies on transcriptional repression by directing DNA cytosine methylation [16] and local histone H3K9 methylation [17] deposition at specific promoter sequences established the feasibility of the approach in the late 1990s and early 2000s. The use of zinc finger-based epigenome editing tools has contributed valuable insights into epigenetic mechanisms, as will be discussed in the next section. However, the high cost and technical expertise required to engineer and validate context-dependent specificity in zinc finger proteins [18, 19] has greatly limited their widespread adoption.

### **2.2. TALE-based systems**

Transcription activator-like effectors (TALEs) are proteins of bacterial origin. They were first reported in the literature in 2007, after two independent research groups discovered that certain bacterial proteins can bind specific promoter sequences in eukaryotic cells [20, 21]. Upon binding to DNA, the pathogenic effector proteins induce the expression of genes that promote the spread of bacterial infection in host cells. TALEs are secreted by gram-negative bacteria of the *Xanthomonas* genus and injected into eukaryotic cells via the type III secretion system [22]. Once inside the eukaryotic cell, they translocate directly to the nucleus where they act as transcription factors to regulate the expression of genes that support bacterial infection [20, 21].

The molecular basis for DNA recognition of each TALE comes from a central tandem amino acid repeat domain of approximately 33–35 residues in length [23, 24]. This tandem repeat is flanked by an N-terminal region required for type III secretion and a C-terminal region involved in nuclear localization and transcriptional activation. Each repeat folds into two left-handed α helices linked by a short loop that contains two hypervariable residues, known as the repeat variable diresidues (RVDs). The RVDs occupy positions 12 and 13 of each repeat, where the 12th residue stabilizes the RVD loop by mediating contacts with the protein backbone, while the 13th residue interacts directly with a specific nucleotide nitrogenous base (**Figure 2**). Thus, the DNA specificity of TALEs is modular and encoded in the tandem repeat sequence [25, 26].

comprises an antiparallel β sheet and α helix, which are stabilized by the zinc ion and a set of hydrophobic residues [11]. The α helix of each zinc finger binds directly to the major groove

base-pair target site [11]. Thus, side chain residues in the α helix that can be engineered to recognize diverse nucleic acid base-pair triplets drive the double-stranded DNA sequence

The intrinsic modularity of DNA recognition by zinc finger proteins led to the swift development of a range of biotechnological applications. For example, manipulation of gene expression via site-specific, DNA-binding zinc finger proteins was first established by targeting a 9 base-pair region of a *BCR*-*ABL* fusion oncogene [12]. Using a three-zinc finger peptide, scientists demonstrated that transcriptional repression of a leukemic oncogene in a chromosomal

Similarly, the development of zinc finger nucleases (ZFNs) marked an important milestone for modern genome editing biotechnologies. ZFNs are engineered chimeric fusions composed of a set of tandem zinc finger DNA-binding proteins and a nuclease domain—such as the catalytic domain of the restriction endonuclease *Fok*I—that cleaves DNA [13]. Synthetic zinc finger-*Fok*I fusions coupled the DNA sequence specificity of zinc finger proteins with the nonspecific cleavage activity of *Fok*I to trigger double-stranded breaks at desired genomic loci. Importantly, repurposing zinc finger proteins into ZFNs facilitated the path for epigenome

editing biotechnologies aimed at controlling transcriptional activation and repression.

Zinc finger-related epigenome editing tools are based on the concept of fusing programmable, DNA binding, zinc finger proteins designed to target diverse sequences [14, 15] with catalytically active or scaffolding effector domains. The chimeric proteins are designed to alter gene expression patterns and act as artificial transcription factors (ATFs) [15]. Pioneer studies on transcriptional repression by directing DNA cytosine methylation [16] and local histone H3K9 methylation [17] deposition at specific promoter sequences established the feasibility of the approach in the late 1990s and early 2000s. The use of zinc finger-based epigenome editing tools has contributed valuable insights into epigenetic mechanisms, as will be discussed in the next section. However, the high cost and technical expertise required to engineer and validate context-dependent specificity in zinc finger proteins [18, 19] has greatly limited their

Transcription activator-like effectors (TALEs) are proteins of bacterial origin. They were first reported in the literature in 2007, after two independent research groups discovered that certain bacterial proteins can bind specific promoter sequences in eukaryotic cells [20, 21]. Upon binding to DNA, the pathogenic effector proteins induce the expression of genes that promote the spread of bacterial infection in host cells. TALEs are secreted by gram-negative bacteria of the *Xanthomonas* genus and injected into eukaryotic cells via the type III secretion system [22]. Once inside the eukaryotic cell, they translocate directly to the nucleus where they act as transcription factors to regulate the expression of genes that support bacterial infection [20, 21].


of B-DNA. Residues from the NH<sup>2</sup>

specificity of each zinc finger.

148 Chromatin and Epigenetics

DNA context was possible [12].

widespread adoption.

**2.2. TALE-based systems**

Unlike zinc fingers, which require triplet sequence recognition sites, TALEs are able to target a single nucleotide at a time through its RVDs. This characteristic makes TALEs simpler to engineer and has enabled the rational design of artificial TALEs for biotechnological applications [26]. Indeed, borrowing from its ZFN predecessors, TALE proteins fused to the *Fok*I nuclease domain gave rise to TALE nucleases (TALENs), which have been utilized for genome editing applications [27].

Similarly, fusions of TALEs to epigenetic effectors have been repurposed for epigenome editing to manipulate gene expression. For example, TALE fusions to activation domains have shown the ability to target specific genomic loci and induce robust transcriptional activation [28]. Inactivation of enhancer regulatory elements has also been demonstrated using TALE fusions to chromatin effectors targeting specific histone modifications [29], thereby establishing a platform to interrogate the function of specific regulatory elements in diverse chromatin landscapes.

**Figure 2. Structure of a TAL effector bound to its target DNA**. (A) The TALE tandem amino acid repeats (multi-colored helices) associate into a right-handed superhelix that wraps around the major groove of the target DNA (blue). (B) Orthogonal view of target DNA recognition by a TALE [PDB 3UGM].

Despite the advantages of possessing modular properties compared to zinc finger proteins and the improvements made in design and assembly of custom TALE-based systems [30, 31], significant technological shortcomings remain to be addressed. The highly repetitive sequences associated with TALE tandem repeats make them susceptible to rearrangements when expressed in target cells due to recombination events that trigger deletions [32]. Moreover, the sheer size of TALE repeats creates obstacles to cellular delivery using some standard viral vectors—e.g., lentiviruses [32]. Such limitations have precluded wider adaptability of TALE-based tools for epigenome editing.

CRISPR–Cas systems are currently classified into class 1 and class 2 groups, which are further divided into six different types and multiple subtypes [36]. Adaptive immunity to foreign nucleic acids is encoded in CRISPR arrays, which contain DNA sequences derived from the integration of invasive DNA as a new CRISPR spacer. CRISPR arrays are transcribed into precursor CRISPR RNAs (pre-crRNAs) and subsequently processed into mature CRISPR RNAs (crRNAs). After processing takes place, crRNAs mediate interference by acting as guides that recruit a variety of Cas effector proteins to complementary nucleic acids from invasive genetic elements [36]. Unlike zinc finger- and TALE-based systems, which rely on protein-DNA interactions to target specific genetic loci, CRISPR–Cas systems are DNA-encoded and RNA-guided. Thus, CRISPR–Cas systems harness nature's principles of Watson-Crick base pairing of nucleic acids to identify the target genetic loci. This property confers significant advantages to CRISPR–Cas systems because

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they bypass the need for complex protein engineering to mediate DNA recognition.

paved the path for repurposing the system for epigenome editing applications.

pact catalytic core [38].

and TALE fusions to epigenetic effectors.

The type II CRISPR–Cas9 system is the most characterized CRISPR–Cas system to date. This complex requires dual RNA molecules—a crRNA and a trans-activating crRNA (tracrRNA) that hybridize together and direct Cas9, a DNA endonuclease, to a target DNA sequence. Upon target recognition, Cas9 triggers double-stranded breaks a few nucleotides away from a protospacer adjacent motif (PAM) site [37]. The RNA-guided Cas9 endonuclease cleaves double-stranded DNA via conserved RuvC and HNH nuclease domains, which form a com-

In recent years, the CRISPR–Cas9 system has been repurposed into an efficient genome editing tool by engineering a chimeric single guide RNA (sgRNA) that fuses the crRNA and tracrRNA into a programmable RNA molecule capable of sequence-specific DNA targeting [37]. Genome editing biotechnological applications take advantage of Cas9-induced doublestranded DNA breaks to mediate DNA repair via one of two pathways: nonhomologous end joining (NHEJ), which introduces insertion or deletion mutations, or homology-directed repair (HDR), which introduces specific mutations or DNA sequences by recombination with a donor template. Importantly, adapting the CRISPR–Cas9 system for gene editing has also

CRISPR-Cas9–mediated epigenome editing is based on the use of an engineered Cas9 protein, which has been purposefully stripped of its catalytic activity into a nuclease-null or "dead" Cas9 (dCas9). Mutating two residues—D10A and H840A—located in the RuvC and HNH nuclease domains is necessary and sufficient to deactivate the nuclease activity of the enzyme [38]. The mutations allow CRISPR–Cas9 to target specific DNA sequences and bind to them without cleaving DNA. Epigenetic effector proteins can then be fused to dCas9 to trigger desired local epigenetic changes, in much the same way mentioned earlier using zinc finger

CRISPR–dCas9 has recently been used to demonstrate the feasibility of targeting specific epigenetic modifications to trigger transcriptional activation or repression. For instance, tethering dCas9 to an epigenetic effector that acetylates histone tail residues led to transcriptional activation in one study [39], and a dCas9 fusion to a repression domain showed the CRISPR– dCas9 system's ability to recruit a heterochromatin-forming complex to silence gene expression [40]. The next section discusses these and other epigenome editing studies in more detail.

### **2.3. CRISPR–Cas–based systems**

Clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated sequences (Cas) constitute bacterial and archaeal endogenous adaptive immunity systems. They were first reported over three decades ago upon publication of findings of a cryptic cluster of unknown function in the bacterium *Escherichia coli* [33]. Advances in bioinformatics alongside greater access to bacterial genomes in public databases around the turn of the century—led to the realization that such clusters exist in a wide range of bacteria and archaea [34]. Interest about the functional significance of the clusters in the scientific community grew steadily, which eventually led researchers to empirical evidence that CRISPR–Cas systems provide adaptive immunity against attack by viruses and plasmids [35] **Figure 3**.

**Figure 3.** Structural representation of CRISPR–Cas9 in complex with target double-stranded DNA and sgRNA. (A) Crystal structure of the CRISPR–Cas9 nuclease (gray) bound to a sgRNA (orange) and double-stranded target DNA (blue) primed for cleavage. Mutation of two Cas9 residues within the RuvC and HNH nuclease domains—D10A and H840A (green spheres)—abolishes the catalytic activity of the enzyme and transforms it into dCas9, which is able to bind its target sequence without triggering DNA cleavage. (B) Schematic cartoon representation of the CRISPR–Cas9– sgRNA–DNA complex (colors as shown in A). [PDB 5F9R].

CRISPR–Cas systems are currently classified into class 1 and class 2 groups, which are further divided into six different types and multiple subtypes [36]. Adaptive immunity to foreign nucleic acids is encoded in CRISPR arrays, which contain DNA sequences derived from the integration of invasive DNA as a new CRISPR spacer. CRISPR arrays are transcribed into precursor CRISPR RNAs (pre-crRNAs) and subsequently processed into mature CRISPR RNAs (crRNAs). After processing takes place, crRNAs mediate interference by acting as guides that recruit a variety of Cas effector proteins to complementary nucleic acids from invasive genetic elements [36]. Unlike zinc finger- and TALE-based systems, which rely on protein-DNA interactions to target specific genetic loci, CRISPR–Cas systems are DNA-encoded and RNA-guided. Thus, CRISPR–Cas systems harness nature's principles of Watson-Crick base pairing of nucleic acids to identify the target genetic loci. This property confers significant advantages to CRISPR–Cas systems because they bypass the need for complex protein engineering to mediate DNA recognition.

Despite the advantages of possessing modular properties compared to zinc finger proteins and the improvements made in design and assembly of custom TALE-based systems [30, 31], significant technological shortcomings remain to be addressed. The highly repetitive sequences associated with TALE tandem repeats make them susceptible to rearrangements when expressed in target cells due to recombination events that trigger deletions [32]. Moreover, the sheer size of TALE repeats creates obstacles to cellular delivery using some standard viral vectors—e.g., lentiviruses [32]. Such limitations have precluded wider adapt-

Clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated sequences (Cas) constitute bacterial and archaeal endogenous adaptive immunity systems. They were first reported over three decades ago upon publication of findings of a cryptic cluster of unknown function in the bacterium *Escherichia coli* [33]. Advances in bioinformatics alongside greater access to bacterial genomes in public databases around the turn of the century—led to the realization that such clusters exist in a wide range of bacteria and archaea [34]. Interest about the functional significance of the clusters in the scientific community grew steadily, which eventually led researchers to empirical evidence that CRISPR–Cas systems

**Figure 3.** Structural representation of CRISPR–Cas9 in complex with target double-stranded DNA and sgRNA. (A) Crystal structure of the CRISPR–Cas9 nuclease (gray) bound to a sgRNA (orange) and double-stranded target DNA (blue) primed for cleavage. Mutation of two Cas9 residues within the RuvC and HNH nuclease domains—D10A and H840A (green spheres)—abolishes the catalytic activity of the enzyme and transforms it into dCas9, which is able to bind its target sequence without triggering DNA cleavage. (B) Schematic cartoon representation of the CRISPR–Cas9–

provide adaptive immunity against attack by viruses and plasmids [35] **Figure 3**.

ability of TALE-based tools for epigenome editing.

sgRNA–DNA complex (colors as shown in A). [PDB 5F9R].

**2.3. CRISPR–Cas–based systems**

150 Chromatin and Epigenetics

The type II CRISPR–Cas9 system is the most characterized CRISPR–Cas system to date. This complex requires dual RNA molecules—a crRNA and a trans-activating crRNA (tracrRNA) that hybridize together and direct Cas9, a DNA endonuclease, to a target DNA sequence. Upon target recognition, Cas9 triggers double-stranded breaks a few nucleotides away from a protospacer adjacent motif (PAM) site [37]. The RNA-guided Cas9 endonuclease cleaves double-stranded DNA via conserved RuvC and HNH nuclease domains, which form a compact catalytic core [38].

In recent years, the CRISPR–Cas9 system has been repurposed into an efficient genome editing tool by engineering a chimeric single guide RNA (sgRNA) that fuses the crRNA and tracrRNA into a programmable RNA molecule capable of sequence-specific DNA targeting [37]. Genome editing biotechnological applications take advantage of Cas9-induced doublestranded DNA breaks to mediate DNA repair via one of two pathways: nonhomologous end joining (NHEJ), which introduces insertion or deletion mutations, or homology-directed repair (HDR), which introduces specific mutations or DNA sequences by recombination with a donor template. Importantly, adapting the CRISPR–Cas9 system for gene editing has also paved the path for repurposing the system for epigenome editing applications.

CRISPR-Cas9–mediated epigenome editing is based on the use of an engineered Cas9 protein, which has been purposefully stripped of its catalytic activity into a nuclease-null or "dead" Cas9 (dCas9). Mutating two residues—D10A and H840A—located in the RuvC and HNH nuclease domains is necessary and sufficient to deactivate the nuclease activity of the enzyme [38]. The mutations allow CRISPR–Cas9 to target specific DNA sequences and bind to them without cleaving DNA. Epigenetic effector proteins can then be fused to dCas9 to trigger desired local epigenetic changes, in much the same way mentioned earlier using zinc finger and TALE fusions to epigenetic effectors.

CRISPR–dCas9 has recently been used to demonstrate the feasibility of targeting specific epigenetic modifications to trigger transcriptional activation or repression. For instance, tethering dCas9 to an epigenetic effector that acetylates histone tail residues led to transcriptional activation in one study [39], and a dCas9 fusion to a repression domain showed the CRISPR– dCas9 system's ability to recruit a heterochromatin-forming complex to silence gene expression [40]. The next section discusses these and other epigenome editing studies in more detail.
