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

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.

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

C), 5-formylcytosine (f<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.

regulatory action to upregulate patterns of gene expression [60–62].

**3.2. Transcriptional repression**

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

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.

C), and 5-carboxylcyto-

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(m<sup>5</sup>

sine (ca<sup>5</sup>

dC) to form 5-hydroxymethylcytosine (hm<sup>5</sup>
