Epigenetic Modification

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.

Mechanisms of Epigenetic Modification

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.

CRISPR-Cas mediated gene regulation and innate immune evasion by F. novicida CRISPR-Cas mediated gene regulation and innate immune evasion by F. novicida

Fig 1. DNA methylation and demethylation mediated by Cas9. (The circled "me" represents methylation at a specific CpG site.)

CRISPR-Cas mediated gene regulation and innate immune evasion by F. novicida

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.

Chemical- and Photo- Inducible Epigenetic Modification

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.

Probing Functional Epigenetic Roles for Non-coding RNA Manipulation

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.

Epigenetic Modification Related References

1. Zhang et al. Development and application of CRISPR-Cas9 technologies in genomic editing. Human Molecular Genetics, 2018;27(R2):R79-R88.
2. Hsu et al. Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell. 2014 June 5; 157(6): 1262–1278.
3. Lo A and Qi L. Genetic and epigenetic control of gene expression by CRISPR-Cas systems. F1000Research 2017, 6(F1000 Faculty Rev):747.
4. Xie et al. Novel Epigenetic Techniques Provided by the CRISPR-Cas9 System. Stem Cells International. Volume 2018, Article ID 7834175, 12 pages.
5. Enríquez. CRISPR-Mediated Epigenome Editing. YALE JOURNAL OF BIOLOGY AND MEDICINE 89 (2016), pp.471-486.