Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
. 2020 Apr 16;9(4):993.
doi: 10.3390/cells9040993.

CRISPR/Cas9 Epigenome Editing Potential for Rare Imprinting Diseases: A Review

Linn Amanda Syding  1 , Petr Nickl  1 , Petr Kasparek  1   2 , Radislav Sedlacek  1   2
Affiliations
Review

CRISPR/Cas9 Epigenome Editing Potential for Rare Imprinting Diseases: A Review

Linn Amanda Syding et al. Cells. .

Abstract

Imprinting diseases (IDs) are rare congenital disorders caused by aberrant dosages of imprinted genes. Rare IDs are comprised by a group of several distinct disorders that share a great deal of homology in terms of genetic etiologies and symptoms. Disruption of genetic or epigenetic mechanisms can cause issues with regulating the expression of imprinted genes, thus leading to disease. Genetic mutations affect the imprinted genes, duplications, deletions, and uniparental disomy (UPD) are reoccurring phenomena causing imprinting diseases. Epigenetic alterations on methylation marks in imprinting control centers (ICRs) also alters the expression patterns and the majority of patients with rare IDs carries intact but either silenced or overexpressed imprinted genes. Canonical CRISPR/Cas9 editing relying on double-stranded DNA break repair has little to offer in terms of therapeutics for rare IDs. Instead CRISPR/Cas9 can be used in a more sophisticated way by targeting the epigenome. Catalytically dead Cas9 (dCas9) tethered with effector enzymes such as DNA de- and methyltransferases and histone code editors in addition to systems such as CRISPRa and CRISPRi have been shown to have high epigenome editing efficiency in eukaryotic cells. This new era of CRISPR epigenome editors could arguably be a game-changer for curing and treating rare IDs by refined activation and silencing of disturbed imprinted gene expression. This review describes major CRISPR-based epigenome editors and points out their potential use in research and therapy of rare imprinting diseases.

Keywords: Angelman syndrome; CRISPR/Cas9; Prader-Willi syndrome; Silver-Russell syndrome; epigenome editing; genomic imprinting; rare disease; transcriptome editing; transient neonatal diabetes mellitus.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Epi-editor systems and their constitution. (A) Cas9 nuclease executing site-specific DSB; (B) dCas9 protein with effector domain of DNMTs or TETs or p300 or PRDM9 or LSD1 or HDAC3. DNMTs repress gene regulation through DNA methylation, TETs mediate demethylation of DNA and activate gene expression. p300 acetylates H3K27 and PRDM9 adds a third methyl residue on H3K4, with both effectors promoting gene expression. LSD1 removes methyl groups from H3K4me1/2 and H3K9me2, and HDAC3 deacetylates H3K27ac, with both modifications leading to repression of gene expression; (C) dCas9 protein with inactivation mutations, D10A and H84A in domain RvuC and HNH, respectively (D); CRISPR activator, dCas9 fused to distinct trans-activation proteins, such as VP64, p65, Rta; (E) CRISPR interference complex, dCas9 with KRAB repressing gene expression (F) CRISPRa, synergistic activation modulator (SAM) tethering trans-activating molecules (p65 and HSF1) on RNA scaffold through MS2 proteins. (G) CRISPRa, gene activating SunTaq system, consisting of dCas9 with repetitive peptide epitopes bound by single-chain variable fragment antibodies (ScFv) fused to trans-activation proteins (p65 and HSF1).
Figure 2
Figure 2
Light inducible CRISPR/Cas9 systems. (A) An inducible system based on blue light-dependent interaction between cytochrome 2 and CIB1 protein fused to CRISPRa components dCas9 and the effector (VP64 or p65). During exposure to the blue light two components are bound together and fully functional, upon removal of the blue light the complex is decomposed; (B) CASANOVA system, the blue light-inducible system controlling Cas9 nuclease activity via inhibitor LOV2-AcrIIA4. In the absence of the blue light, the inhibitor blocks Cas9 and prevents it from the binding the target sequence. In the presence of the blue light, the inhibitor is destabilized and released from Cas9 protein. Subsequently, Cas9 is active and executes DSB in the target locus when the blue light is removed the inhibitor binds back to Cas9.The chemically inducible systems are ligand-dependent. The interaction between the effector domain and dCas9 is conditioned by the presence of a ligand and two ligand-binding domains, linking two epi-editor components. Again, one of the binding domains fuses with the effector and the other with dCas9. In the presence of a ligand, both binding domains interact with the ligand and form a stable heterodimer resulting in the formation of an active epi-editor complex. Examples of ligand-binding domains are FK506 binding protein 12 (FKBP), and FKBP rapamycin binding protein (FRB) interacting together via rapamycin molecule (Figure 3A) [100], abscisic acid-induced dimerization of ABI and PYL1 domains (Figure 3B) [101], or gibberellin-induced dimerization of GID1 and GAI (Figure 3B) [101].
Figure 3
Figure 3
Chemically Inducible Epi-editors Systems. (A) Split dCas9-VP64 complex with one ligand-binding domain (LBD) at N-terminus of dCas9 and second LBD at C-terminus. Both LBDs bind a ligand (rapamycin, yellow) and bring together both halves of dCas9, resulting in the formation of functional gene activation complex; (B) Inducible system using phytohormones and phytohormone binding domains ABI or GAI fused to dCas9, and PYL1/GID1 fused to the effector-activator (VPR) or KRAB. The interaction via a ligand (abscisic acid or gibberellin) activates the epi-editor complex; (C) Inducible SAM system with a destabilized domain of estrogen receptor 50 (ER50DD). In the absence of a ligand-4OHT (4-hydroxytamoxifen), ER50DD protein is destabilized and leads the whole ER50DD-MS2-p65-HSF1 to proteasomal degradation, once the ligand is present it binds ER50DD and stabilizes it. Then complex is not degraded and therefore capable of interacting with RNA aptamers a form functional activation complex. (D) Inducible system with proteolytic cleavage. A part of the linker, between dCas9 and effector domain, is NS3 protein, protease from hepatitis C virus that cleaves peptide bonds in its vicinity. When NS3 is active, it cleaves the linked and abrogates the function of the epi-editor complex. The protease can be blocked by inhibitorBLIN-2061, leading to restoration of the epi-editor and its activity. The effect of ligands or inhibitors in the system mentioned above is reversible. After the inhibitor/ligand is diluted or metabolized, the chemical epi-editor systems are inactivated.
Figure 4
Figure 4
Schematic of the AS/PWS locus. The pink filled boxes: paternally expressed genes, blue filled boxes: maternally-expressed genes. The PWS/IC located in the promoter/exon 1 region of SNRPN/SNURF is hypermethylated on the maternally inherited chromosome thus silencing transcription of the Ube3a-ATS, allowing Ube3a to be expressed. The paternally-inherited PWS-IC is hypomethylated thus expressing the transcript, silencing Ube3a.
Figure 5
Figure 5
Schematic of the disease causes for TNDM. Three etiologies are depicted, paternal UPD of chromosome 6, paternal duplication of chromosome 6, and hypomethylation on the maternal HYMA1/PLAGL1 promoter, thus allowing for biallelic expression.
Figure 6
Figure 6
Locus overview of the SRS region. The upper drawing depicts the maternal regulation of the locus. The ICR2 is hypermethylated where the maternally expressed genes KCNQ1 and CDKNIC are expressed. The ICR1 is not methylated, allowing the CTCF motif to bind it and hindering the enhancers (E) to activate IGF2 expression. The lower drawing paternally inherited ICR1 is methylated, thus inhibiting CTCF binding and allowing the enhancers to regulate IGF2 transcription. The ICR2 is not methylated paternally and the non-coding transcript KCNQ1OTI is expressed. Adapted from: Azzi et al. (2009) [158].
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 2012;337:816–821. doi: 10.1126/science.1225829. - DOI - PMC - PubMed
    1. Jinek M., Jiang F., Taylor D.W., Sternberg S.H., Kaya E., Ma E., Anders C., Hauer M., Zhou K., Lin S., et al. Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation. Science. 2014;343:1247997. doi: 10.1126/science.1247997. - DOI - PMC - PubMed
    1. Gilbert L.A., Horlbeck M.A., Adamson B., Villalta J.E., Chen Y., Whitehead E.H., Guimaraes C., Panning B., Ploegh H.L., Bassik M.C., et al. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell. 2014;159:647–661. doi: 10.1016/j.cell.2014.09.029. - DOI - PMC - PubMed
    1. Kato T., Hara S., Goto Y., Ogawa Y., Okayasu H., Kubota S., Tamano M., Terao M., Takada S. Creation of mutant mice with megabase-sized deletions containing custom-designed breakpoints by means of the CRISPR/Cas9 system. Sci. Rep. 2017;7:59. doi: 10.1038/s41598-017-00140-9. - DOI - PMC - PubMed
    1. Kitamoto K., Taketani Y., Fujii W., Inamochi A., Toyono T., Miyai T., Yamagami S., Kuroda M., Usui T., Ouchi Y. Generation of mouse model of TGFBI-R124C corneal dystrophy using CRISPR/Cas9-mediated homology-directed repair. Sci. Rep. 2020;10:1–10. doi: 10.1038/s41598-020-58876-w. - DOI - PMC - PubMed

Publication types

MeSH terms

Supplementary concepts