CRISPR-Based Epigenome Editing: Mechanisms and Applications

Figures & data

Figure 1. Molecular mechanisms modulating the epigenome.

The epigenome encompasses a variety of dynamic molecular processes crucial for cell function and development. (A) Nucleosomes, composed of DNA (black line) wrapped around histones (blue) are the basic units of chromatin packaging in eukaryotes. Histones tails can be modified by the covalent additions of chemical groups catalyzed by enzymes (colored circles), ultimately effecting chromatin accessibility. (B) Methyltransferases may add a methyl group to the C-5 position of cytosine, which may affect transcription status. (C) Noncoding RNA elements such as lncRNAs (red line) can modulate gene expression by binding TFs (yellow circle). (D) Chromatin is intricately folded into a 3D configuration in the nucleus, constituting chromatin domains and interactions of regulatory elements that affect gene expression.

lncRNA: Long noncoding RNA; TF: Transcription factor.

Table 1. Types of CRISPR systems that have been repurposed for epigenome editing.

Type	I	II	III	IV	V	VI	Ref.
Class	1 (multisubunit)	2 (single unit)	1 (multisubunit)	1 (multisubunit)	2 (single unit)	2 (single unit)
Endonuclease protein	Cas3	Cas9	Cas10	Scf1	Cas12	Cas13
Target molecule	DNA	DNA	DNA/RNA	–	DNA	RNA
Epigenome editing platforms	–	dCas9-DNMT3A dCas9-SunTag-DNMT3A dCas9-DNMT3A-DNMT3L dCas9-DNMT3A-DNMT3L-KRAB dCas9-TET1CD dCas9-Tet1-CD and MS2-Tet1-CD dual system dCas9-TET1 TET1-dSaCas9 and VPR-dSpCas9 dCas9-SunTag-TET1 dCas9-p300 dCas9-HDAC3 dCas9-KRAB dCas9-LSD1 dCas9-Ezh2 dCas9-VPR (CRISPRa)	DiCas7-11	–	–	CRISPR-Cas13d dCas13b-ADAR2 CRISPR-Cas13a	[36,46,47,49,53–76]

Table 2. dCas9-mediated epigenome editing involves multiple epigenetic mechanisms.

Modification	Effector domains	Target	Mechanism	Ref.
DNA methylation	dCas9-DNMT3A dCas9-SunTag-DNMT3A dCas9-DNMT3A-DNMT3L dCas9-DNMT3A-DNMT3L-KRAB	BDNF promoter MyoD enhancer CTCF loops CDKN2A promoter, ARF promoter, Cdkn1a promoter IL6ST, BACH2 HOXA5 EpCAM, CXCR4 and TFRC promoters UNC5C promoter GAPDH-Snrpn reporter system	Targeting of dCas9-DNMT3a fusion proteins to unmethylated promoter sequences Targeting of the dCas9-DNMT3a fusion domain along with repetitive peptide epitopes (SunTag) that can recruit and amplify the local concentration of DNMT3a domains for robust methylation of target loci Targeting of a dCas9-DNMT3A-DNMT3L methyltransferase chimeric construct to induce higher methylation efficiency compared with a system that utilizes a single methyltransferase catalytic domain Development of ‘CRISPRoff’ composed of dCas9-DNMT3A-DNMT3L-KRAB fusion domains to target and induce gene silencing memory	[46,49,56–59,62,77]
DNA demethylation	dCas9-TET1CD dCas9-Tet1-CD and MS2-Tet1-CD dual system dCas9-TET1 TET1-dSaCas9 and VPR-dSpCas9 dCas9-SunTag-TET1 enzyme free steric interference with DNA methyltransferases dCas9-TETv4 with sgRNA modified with MCP (VP64, p65-AD and Rta)	STAT3 binding site upstream of GFAP BRCA1 gene promoter RANKL, MAGEB2, MMP2 neighboring CpGs BDNF promoter MyoD enhancer Dazl-Snrpn-GFP reporter HNF1A, MGAT3 FMR1, SerpinB5, Tnf CLTA	Targeted DNA demethylation using a dCas9 fusion construct with a TET1CD catalytic domain Simultaneous transfection of dCas9-TET1-CD and a MS2 coat protein fusion construct guided with a sgRNA2.0 transcription system to demethylate target genomic regions and promote gene expression Induction of de novo demethylation of target gene regions with the dCas9-TET1 construct Demethylation of regulatory regions via dual TET1-dSaCas9 and VPR-dSpCas9 fusion constructs for synergistic modulation of gene expression Targeting of the dCas9-TET1 fusion domain along with repetitive peptide epitopes (SunTag) that can recruit and amplify the local concentration of TET1 proteins for robust demethylation of target loci Targeting of the enzyme-free dCas9 system to CpGs adjacent to transcription start sites to interfere with the binding of DNA methyltransferases leading to induced site-specific gene expression Development of ‘CRISPRon’ composed of a dCas9-TETv4 fusion domain with modified sgRNAs with MCP and various combinations of transactivation domains to reverse ‘CRISPRoff’-mediated gene silencing	[46,53–57,60,61,77]
H3K27 acetylation	dCas9-p300 dCas9-p300 fused with PYL or ABI proteins dLbCpf1-P300/dCpf1-SunTag dCas9-p300 and MCP-VP64 dual system	IL1RN (MYOD), POU5F1/OCT4 IL1RN GRM2 MYOD, HS2 enhancer, TAL1 IL1RN, GRM2, HBA, MYOD IL1RN, RHOXF2, TTN	Targeting of dCas9-p300 acetyltransferase domain to promoters and distal enhancers leading to gene activation Utilizing CIP for temporal control of histone modifications, where p300 and dCas9 proteins can be fused to ABI and PYL that can dimerize when the small chemical inducer ABA is introduced to reversibly recruit p300 to target sites System based on a Cpf1 and p300 fusion domain for targeted histone acetylation as an alternative to dCas9 to activate multiple genes with a single sgRNA enhancer targeting by ‘enCRISPRa’ system for activation	[47,63–65,78–82]
H3K27 deacetylation	dCas9-HDAC3 dCas9-KRAB	Smn1, Mecp2, Isl1 OCT4, Tbx3, SOX2	Targeting of the dCas9-HDAC3 deacetylase domain to promoters and enhancers leading to a repressive state Targeting of the dCas9-KRAB deacetylase domain to promoters and enhancers leading to a repressive state	[66,67]
H3K4 demethylation	dCas9-LSD1	OCT4, Tbx3, SOX2	Targeting of the dCas9-LSD1 demethylase domain to promoters and enhancers leading to activation	[67]
H3K4 methylation	dCas9-SET	ICAM1, RASSF1a, EpCAM IL1RN, GRM2	Targeting of the dCas9-SET histone methyltransferase domain to promoters leading to gene reactivation	[63,83]
H3K9 methylation	dCas9-G9A[SET]/dCas9-SUV[SET] dCas9-KRAB	HER2, MYC, EPCAM HS2 enhancer	Repression of target genes by histone methyltransferases fusion domains with dCas9 Targeting of dCas9-KRAB to enhancer regions and recruitment of repressive chromatin methylation modifiers	[68,84]
H3K27 methylation	dCas9-olEzh2 dCas9-Ezh2	Arhgap35, Pfkfb4a, Nanos3, Dcx, Kita, Tbx16, Slc41a2a HER2, MYC, EPCAM C/ebpa promoter	Repression of target genes by histone methyltransferases fusion domains with dCas9	[68,69,85]
RNA	DECKO (double-excision CRISPR knockout) CRISPR-Cas13d dCas9-VPR (CRISPRa) dCas13b-ADAR2 DiCas7-11 CRISPR-Cas13a	UCA1, MALAT1, TFRC mCherry, B4GALNT1, ANXA4, HOTTIP, MALAT1, EGFR, EZH2, HRAS, KRAS, NF2, NFKB1, NRAS, PPARG, RAF1, SMARCA4, STAT3 vlincRNAs (ID30, ID332, ID-372, ID-604) RP11-326A19.4 Cluc W85X Gluc/Cluc luciferases PPIB, KRAS, MALAT1 CXCR4 RFP Trans-ssRNA	Knock out of long noncoding RNA genes by the deletion of promoters Programmable RNA targeting and cleavage Targeted knockdown of vlincRNAs Activation of long noncoding RNA promoter region inducing genome wide transcriptional changes ADAR2 domain fusion with dCas13 for targeted RNA editing through the direct adenosine to inosine deaminase activity of ADAR2 Engineering of Cas7-11 for RNA targeting and knockdown Guided cleavage of ssRNAs by CRISPR-Cas13a effector	[36,70–76,86]
TF hinderance	CRISPR-dCas9	OCT4, Nanog, Pax6	By Steric hinderance of transcription factors by dCas9 targeted binding leading to disturbed TF-DNA interaction	[87]
Genome organization	dCas9-DNMT3A dCas9 fused with PYL or ABI proteins (CLOuD9) dCas9-CIBN	miR290, Pou5f1 β-globin promoter, LCR HS2, OCT4 Klf4, Zfp462 promoter	Targeted methylation of CTCF anchor sites blocking CTCF binding and altering DNA looping and expression of genes located in neighboring loops Utilizing CIP for selective formation of de novo chromatin loops dCas9 proteins can be fused to ABI and PYL that can dimerize when the small chemical inducer ABA is introduced to reversibly bring two genomic regions into juxtaposition LADL system that drives de novo chromatin looping by the heterodimerization of dCas9-CIBN fusion proteins when blue light is applied using cryptochrome 2 inducible bridging factor	[56,88,89]

CIP: Chemically induced proximity; LADL: Light-activated dynamic looping; MCP: MS2 coat protein; ssRNA: Single-stranded RNA; TF: Transcription factor; vlincRNA: Very long intergenic noncoding RNA.

Figure 2. Epigenome and RNA editing via CRISPR.

(A) Cas protein (light blue) binds DNA target sequence (orange) via guide RNA (orange line) near protospacer adjacent motif (PAM) (blue). This leads to cleavage of the target sequence due to nuclease activity of the Cas protein, producing double-stranded breaks that will be mend by the endogenous DNA repair machinery. (B) dCas lacks the ability to induce breaks in target sequences. However, can still bind to target sequences via gRNAs and add/remove specific epigenetic marks or modulate chromosome looping. (C) Cas13, guided by gRNA, targets the RNA (yellow) molecule for cleavage. (D) However, dCas13 juxtaposed with ADAR2 carries out RNA editing by converting A to I.

ADAR: Adenosine deaminase acting on RNA; dCas9: Nuclease-defective Cas9; gRNA: Guide RNA.

Table 3. Applications of CRISPR mediated epigenome editing in cancer research and regenerative medicine.

Application	Target	Mechanism	Ref.
Cancer therapeutics	GRN in Hep3B hepatoma cells SARI promoter in colon cancer tissue DKK3 promoter in PCa cells ΔNp63 in in lung and esophageal SCCs Two class II TSGs, MASPIN, REPRIMO in H157 lung cancer cells and AGS gastric cancer cells MDM2 in osteosarcoma cells KLF4 in UBC Rnd3 in MM RPMI 8226 and JJN3 cell lines	Targeting of a dCas9 fusion system with DNMT3a, EZH2 and KRAB epi-suppressors to the GRN promoter, leading to knockdown and reduction of tumor formation Targeted demethylation of the SARI promoter by a dCas9 TET1 fusion system, leading to restoring SARI antitumor function Targeted demethylation of the DKK3 promoter by a dCas9-VPR fusion system, leading to induction of DKK3 tumor suppressive roles CRISPR interference system (dCas9-KRAB) targeted to the transcription start site of ΔNp63, leading its suppression and induced apoptosis of cancer cells CRISPR/dCas9 fused with a number of effector domains (VP64, p300, VPR, SAM complex) for dormant tumor suppressor gene reactivation to inhibit cell proliferation Control of the MDM2 proto-oncogene by dCas9-KRAB in osteosarcoma cells, leading to effective inhibition of tumor growth Upregulation of KLF4 expression by dCas9-VP64 (CRISPRon), leading to the inhibition of tumorigenesis Silencing of Rnd3 in multiple myeloma cells by dCas9-KRAB	[96,135–145]
Regenerative medicine	UCP1 in adipose-derived stem cells (ADSCs) Pparγ2, Prdm16, Zfp423, Ucp1 in adipocytes Ascl1, Lmx1a, Nr4a2 (ALN) or Ascl1, Lmx1a, NeuroD1 transcription factors FMR1 CGG expansion in induced pluripotent stem cells Brn2, Ascl1, Myt1l in fibroblasts	Upregulation of UCP1 expression in engineered adipocytes using a CRISPR activation system (CRISPRa) composed of dCas9 and activation domains MCP-p65-HSF1 Upregulation of genes involved in adipocyte differentiation and function by the CRIPSRa SAM system for adipocyte engineering Reprogramming of striatal astrocytes into mature neurons by the CRISPRa system dCas9-VP64/SAM for voluntary motor behavior rescue in Parkinson’s disease Targeted demethylation and reactivation of FMR1 by dCas9-TET1 Reprogramming mouse embryonic fibroblasts to induced neuronal cells utilizing dCas9-VP64 to activate endogenous neurogenic genes Brn2, Ascl1, Myt1l	[146–154]

GRN: Granulin; MM: Multiple myeloma; PCa: Prostate cancer; SAM: Synergistic activation mediator; SCC: Squamous cell carcinoma; UBC: Urothelial bladder cancer.

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