**4. Conclusion**

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

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

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,

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

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

and CRISPR–dCas9 proteins at target promoter and enhancer regions.

elements.

154 Chromatin and Epigenetics

repression than single SID domain fusions [66].

synthetic gene repression [62, 72].

**3.3. Technological limitations**

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 biotechnology in the near future.
