What are the applications of CRISPR/Cas9-mediated Epigenetic Editing? The CRISPR/Cas9 with the advantages of low cost and ease of use gives researchers the opportunity to manipulate the epigenome and observe its possible effects on cell function, development, and differentiation (Figure 1). Using non-active dCas9 (another type of Cas9 lacking nuclease activity but retaining DNA binding activity) fused enzymes such as DNA methylase, histone acetyltransferase, and deacetylase can be targeted to alter the epigenetic state of precise locations within the genome.

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Applications:

- silence gene expression of target genes

- activate gene expression of target genes

- determine the role of DNA methylation and histone modifications (methylation, acetylation, etc) on specific regulatory sites (e.g., enhancer sequences)

Explanation:

The CRISPR/Cas9 system can be repurposed to edit the epigenome of living cells. For this purpose, it is required 1-to fuse a catalytically inactive 'dead' Cas9 enzyme with enzymes involved in epigenetic mechanisms (e.g., DNA methyltransferases, histone deacetylases, etc) capable of activating or deactivating gene expression, and 2-to design single-guide RNAs (sgRNAs) complementary to the sequence of interest (i.e., target sequences whose epigenome landscape we are trying to modify). For example, dCas9 gene has been fused with the DNA methyltransferase 3a (Dnmt3a) gene to create a dCas9-DNMT3A complex which can be combined with a specific sgRNA in order to study the effects of DNA methylation at a particular gene. These CRISPR/Cas9-mediated epigenome editing systems have shown to be very useful to understand how epigenome drives chromatin folding and dynamics at specific genomic sites (loci), and thus regulate gene expression at the transcriptional level.

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