In recent years, novel epigenetic modification techniques utilizing the CRISPR-Cas9 system are emerged, including epigenome editing, temporal and spatial control of epigenetic e-ectors, non-coding RNA manipulation, chromatin in vivo imaging, and epigenetic element screening. Compared with the previous epigenetic modification technologies ZFN and TALEN, the CRISPR-Cas9 method has some advantages such as its cost-e-ectiveness and easy-manipulating. Instead of redesigning amino acid sequences and synthesizing new DNA-binding proteins, what researchers need to do in the CRISPR-Cas9 system is redesign the programmable guide RNA (sgRNA) sequences and synthesize a new expression cassette. Thanks to the advent of the novel CRISPR-Cas9 technology, the ﬁeld of epigenetic modification begins to thrive.
Main applications of CRISPR-dCas9 system to study and manipulate the epigenome are based on the feasibility to allocate chromatin modiﬁers and ﬂuorescent molecules in a very precise way. This includes the targeted reposition of transcriptional regulators, histone modiﬁers, enzymes responsible for changes in DNA methylation, chromatin-interacting ncRNAs, and ﬂuorescent molecules. To achieve CRISPR-Cas9-mediated epigenetic modification, the main strategy is fusing the Cas9 with a transcription repressor or activator domain, which was known as an epigenetic e-ector (epie-ector). To be speciﬁc, the adaption is inactivating the Cas9 nuclease (dCas9) ﬁrst and further fusing it with an epie-ector domain. Accumulating evidence has proved that this dCas9-epie-ector fusion complex is an e-cient epigenome editing tool.
By utilizing epigenome modifying repressors, including Lys-speciﬁc histone demethylase 1 (LSD1), histone deacetylase (HDAC), DNA methyltransferases DNMT3A and MQ1, and mSin3 interaction domains, the scope of applying CRISPR repression has been extended to epigenetic editing. Similarly, epigenetic modification approaches can also be used for targeted transcriptional activation, such as dCas9 fused with a DNA demethylase or a histone acetyltransferase. Recently, Klann et al. developed a CRISPR-Cas9-based epigenomic regulatory element screening (CERES) system, which combines dCas9-p300Core with dCas9-KRAB to obtain both gain and loss of function information by targeting the same regions with a repressor and an activator.
Fig 1. DNA methylation and demethylation mediated by Cas9. (The circled "me" represents methylation at a speciﬁc CpG site.)
Fig 2. Schematic of temporal and spatial control of epigenome editing.
For example, the dCas9-DNMT3A (DNA methyltransferase 3A) complex was proved to be able to induce methylation at targeted CpG sites within multiple gene promoters. The highest methylation rate was estimated to be 50%. Fusion of the catalytic domain of DNA demethylase TET1 to dCas9 can induce targeted DNA demethylation. The dCas9-TET complex has been shown to be able to rescue epigenetically silenced gene via inducing demethylation at the targeted region in a B2Mtd Tomato K-562 cell line. The dCas9-TET1 fusion can demethylate 30 to 60% of the CpG islands at tested promoters, which drives the transcriptional activation of target genes in various cell types including embryonic stem cells, cancer cell lines, and primary neurons. Transient expression of dCas9-TET and guide RNA together leads to a long-term reactivation eﬀect highly speciﬁcally.
Eﬀorts have been made to improve epigenetic modification eﬃciency. One strategy is incorporating SunTag into the dCas9-epieﬀector complex. SunTag is a repeating peptide array that can simultaneously bind with multiple copies of a certain protein. Several studies have highlighted the utility of the dCas9-SunTag system in improving transactivation robustness. SunTag could also be fused with dCas9-DNMT3A complex to augment CpG methylation at targeted loci. In conclusion, the dCas9-epieﬀector complex could achieve methylation and demethylation at DNA level, rewriting histone marks by inducing methylation or acetylation at nucleosome level, and be optimized to improve the editing eﬃciency.
The dCas9 DNA-binding domain paired with optical-inducible proteins could be utilized to recruit the epieﬀector domain to the targeted DNA site in an inducible and reversible manner. In a light-activated CRISPR-Cas9 eﬀector (LACE) system, CRY2 and CIB1 were fused to transactivation domain VP64 and catalytically deactivated Cas9, respectively. Cotransfection of these fusion protein pairs with guide RNA resulted in detectable levels of gene activation in the presence of blue light. Interestingly, when the N-terminal fragment of CIB1 was fused to both N- and C-terminus of dCas9, gene activation level in response to blue light was signiﬁcantly increased, which is consistent with previous observation that simultaneous recruitment of VP64 domains to the target site had a synergistic eﬀect on gene activation.
Another strategy is to split Cas9 into fragments and further tether them with optical inducible protein pairs. Upon light stimulation, Cas9 fragments would be brought together via light-induced dimerization to reconstruct nuclease activity. Interestingly, in a photo-activatable CRISPR-Cas9 system, when split Cas9 fragments were fused with the CRY2-CIB1 pair, no light-induced Cas9 activity was induced. The nuclease activity was successfully reconstructed upon light stimulation when Cas9 fragments were tethered with positive and negative magnets, respectively. In addition to the CRY2-CIB1protein pair and magnets, the split Cas9 could also be bound with other ligand-binding protein pairs, which would be brought together by diﬀerent chemicals, to achieve multiplexed genome or epigenome regulation simultaneously and rapidly.
Epigenetic modification can now be used to probe functional roles for non-coding RNAs (ncRNAs) involved in gene regulation and epigenetic chromatin dynamics. Few previous studies demonstrate the feasibility of using CRISPR systems to probe ncRNA function by repurposing the sgRNAs that bind dCas9 as scaffolding molecules to trigger loci- specific regulation. Protein-binding cassettes, RNA aptamers, and long ncRNAs at least 4.8 kb long have been used to build CRISPR-Cas9 complexes that may enable precise ectopic targeting of functional RNAs and ribonucleoprotein complexes to specific genomic loci. Incorporation of protein-binding motifs and ncRNAs directly into the stem-loop structures of sgRNAs-at the 5' or 3' positions-is likely to usher in a long-awaited era of ncRNA functional characterization relevant to chromatin regulation.
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