Novel Epigenetic Techniques Provided by the CRISPR/Cas9 System

Abstract

Epigenetics classically refers to the inheritable changes of hereditary information without perturbing DNA sequences. Understanding mechanisms of how epigenetic factors contribute to inheritable phenotype changes and cell identity will pave the way for us to understand diverse biological processes. In recent years, the emergence of CRISPR/Cas9 technology has provided us with new routes to the epigenetic field. In this review, novel epigenetic techniques utilizing the CRISPR/Cas9 system are the main contents to be discussed, including epigenome editing, temporal and spatial control of epigenetic effectors, noncoding RNA manipulation, chromatin in vivo imaging, and epigenetic element screening.

Figure 1

DNA methylation and demethylation mediated by Cas9. DNA sequences are represented by the blue lines. PAM sequences are highlighted by red color. Guide RNA sequences are shown in purple color. DNA polymerase II (PoI II) is represented by the green irregular shape. The circled “me” represents methylation at a specific CpG site. Basically, dCas9 functions as a DNA-binding protein. DNMT3A or TET is the epieffector. The dCas9-epieffector complex is guided to the DNA target by guide RNA via Watson-Crick base pairing to execute (a) DNA methylation or (b) demethylation, thus inducing decreased or increased gene expression.

Figure 2

Histone acetylation and methylation mediated by Cas9. The empty circle attached to the nucleosome represents a specific amino acid of the histone side chain. The circled “acetyl” and “me” represents acetylation and methylation of amino acids, respectively. The dCas9-epieffector complex could be guided to a selected DNA target to achieve (a) acetylation or (b) methylation at histone level to regulate gene expression.

Figure 3

Schematic of temporal and spatial control of epigenome editing. The semicircles labeled with (A) and (B) represent a protein pair. The magenta comb-like lines represent guide RNA sequences. (A) is bound to dCas9, which would be directed by the guide RNA to the DNA target. (B) is bound to the epieffector. (a) Upon stimulation of light or chemicals, (A and B) would pair with each other thus bringing the dCas9 and epieffector together to achieve site-specific epigenome editing at a given time point. (b) Another strategy is splitting the dCas9 into two parts, each of which is bound by protein (A) or (B). Upon light or chemical stimulation, (A) and (B) would gather together to reconstruct an intact dCas9-epieffector complex to achieve site-specific epigenome editing at a given time point.

Figure 4

In vivo imaging of chromatin interaction mediated by Cas9. Guide RNAs direct dCas9 and fluorescent proteins to bind with selected DNA targets, forming the dCas9-gRNA-fluorescent dye complex. At chromosomal level, spatial position changes between chromosomal regions could be reflected by the (a) seperation or (b) interaction of the dual-color fluorescent signals.