A CRISPR-based Approach for Targeted DNA Demethylation

Overview

Journal Cell Discov

Date 2016 Jul 28

PMID 27462456

Citations 180

Authors

Xingxing Xu

Yonghui Tao

Xiaobo Gao

Lei Zhang

Xufang Li

Weiguo Zou

Kangcheng Ruan

Feng Wang

Guo-Liang Xu

Ronggui Hu

Affiliations

Soon will be listed here.

Abstract

In mammalian cells, DNA methylation critically regulates gene expression and thus has pivotal roles in myriad of physiological and pathological processes. Here we report a novel method for targeted DNA demethylation using the widely used clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system. Initially, modified single guide RNAs (sgRNAs) (sgRNA2.0) were constructed by inserting two copies of bacteriophage MS2 RNA elements into the conventional sgRNAs, which would facilitate the tethering of the Tet1 catalytic domain (Tet-CD), in fusion with dCas9 or MS2 coat proteins, to the targeted gene loci. Subsequently, such system was shown to significantly upregulate transcription of the target genes, including RANKL, MAGEB2 or MMP2, which was in close correlation to DNA demethylation of their neighboring CpGs in the promoters. In addition, the dCas9/sgRNA2.0-directed demethylation system appeared to afford efficient demethylation of the target genes with tenuous off-target effects. Applications of this system would not only help us understand mechanistically how DNA methylation might regulate gene expression in specific contexts, but also enable control of gene expression and functionality with potential clinical benefits.

Citing Articles

DNA methylation affects gene expression but not global chromatin structure in .

Morgan W, Amemiya H, Freddolino L bioRxiv. 2025; .

PMID: 39829790 PMC: 11741368. DOI: 10.1101/2025.01.06.631547.

Epigenetics and suicidal behavior in adolescents: a critical review.

Funahashi Y, Dwivedi Y Epigenomics. 2025; 17(4):247-262.

PMID: 39819344 PMC: 11853622. DOI: 10.1080/17501911.2025.2453415.

Targeting the epigenome with advanced delivery strategies for epigenetic modulators.

Guha S, Jagadeesan Y, Pandey M, Mittal A, Chitkara D Bioeng Transl Med. 2025; 10(1):e10710.

PMID: 39801754 PMC: 11711227. DOI: 10.1002/btm2.10710.

CRISPR-Hybrid: A CRISPR-Mediated Intracellular Directed Evolution Platform for RNA Aptamers.

Su-Tobon Q, Fan J, Goldstein M, Feeney K, Ren H, Autissier P Nat Commun. 2025; 16(1):595.

PMID: 39799111 PMC: 11724954. DOI: 10.1038/s41467-025-55957-0.

Targeted demethylation of cathepsin D via epigenome editing rescues pathology in Alzheimer's disease mouse model.

Park M, Ryu H, Heo S, Kim B, Park J, Lim K Theranostics. 2025; 15(2):428-438.

PMID: 39744681 PMC: 11671390. DOI: 10.7150/thno.103455.

References

Robertson K . DNA methylation and human disease. Nat Rev Genet. 2005; 6(8):597-610. DOI: 10.1038/nrg1655. View

Mali P, Aach J, Stranges P, Esvelt K, Moosburner M, Kosuri S . CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol. 2013; 31(9):833-8. PMC: 3818127. DOI: 10.1038/nbt.2675. View

He Y, Li B, Li Z, Liu P, Wang Y, Tang Q . Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science. 2011; 333(6047):1303-7. PMC: 3462231. DOI: 10.1126/science.1210944. View

Duheron V, Hess E, Duval M, Decossas M, Castaneda B, Klopper J . Receptor activator of NF-kappaB (RANK) stimulates the proliferation of epithelial cells of the epidermo-pilosebaceous unit. Proc Natl Acad Sci U S A. 2011; 108(13):5342-7. PMC: 3069202. DOI: 10.1073/pnas.1013054108. View

Milutinovic S, DAlessio A, Detich N, Szyf M . Valproate induces widespread epigenetic reprogramming which involves demethylation of specific genes. Carcinogenesis. 2006; 28(3):560-71. DOI: 10.1093/carcin/bgl167. View

Dion V, Wilson J . Instability and chromatin structure of expanded trinucleotide repeats. Trends Genet. 2009; 25(7):288-97. PMC: 3671858. DOI: 10.1016/j.tig.2009.04.007. View

Konermann S, Brigham M, Trevino A, Hsu P, Heidenreich M, Cong L . Optical control of mammalian endogenous transcription and epigenetic states. Nature. 2013; 500(7463):472-476. PMC: 3856241. DOI: 10.1038/nature12466. View

Hall D, Struhl K . The VP16 activation domain interacts with multiple transcriptional components as determined by protein-protein cross-linking in vivo. J Biol Chem. 2002; 277(48):46043-50. DOI: 10.1074/jbc.M208911200. View

Schmittgen T, Livak K . Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008; 3(6):1101-8. DOI: 10.1038/nprot.2008.73. View

10.

Ito S, Shen L, Dai Q, Wu S, Collins L, Swenberg J . Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science. 2011; 333(6047):1300-3. PMC: 3495246. DOI: 10.1126/science.1210597. View

11.

Maeder M, Linder S, Cascio V, Fu Y, Ho Q, Joung J . CRISPR RNA-guided activation of endogenous human genes. Nat Methods. 2013; 10(10):977-9. PMC: 3794058. DOI: 10.1038/nmeth.2598. View

12.

Krueger F, Kreck B, Franke A, Andrews S . DNA methylome analysis using short bisulfite sequencing data. Nat Methods. 2012; 9(2):145-51. DOI: 10.1038/nmeth.1828. View

13.

Mendenhall E, Williamson K, Reyon D, Zou J, Ram O, Joung J . Locus-specific editing of histone modifications at endogenous enhancers. Nat Biotechnol. 2013; 31(12):1133-6. PMC: 3858395. DOI: 10.1038/nbt.2701. View

14.

Wu H, Zhang Y . Reversing DNA methylation: mechanisms, genomics, and biological functions. Cell. 2014; 156(1-2):45-68. PMC: 3938284. DOI: 10.1016/j.cell.2013.12.019. View

15.

Ito S, DAlessio A, Taranova O, Hong K, Sowers L, Zhang Y . Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature. 2010; 466(7310):1129-33. PMC: 3491567. DOI: 10.1038/nature09303. View

16.

Hsu P, Scott D, Weinstein J, Ran F, Konermann S, Agarwala V . DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013; 31(9):827-32. PMC: 3969858. DOI: 10.1038/nbt.2647. View

17.

Mali P, Esvelt K, Church G . Cas9 as a versatile tool for engineering biology. Nat Methods. 2013; 10(10):957-63. PMC: 4051438. DOI: 10.1038/nmeth.2649. View

18.

Wu S, Zhang Y . Active DNA demethylation: many roads lead to Rome. Nat Rev Mol Cell Biol. 2010; 11(9):607-20. PMC: 3711520. DOI: 10.1038/nrm2950. View

19.

Maeder M, Angstman J, Richardson M, Linder S, Cascio V, Tsai S . Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins. Nat Biotechnol. 2013; 31(12):1137-42. PMC: 3858462. DOI: 10.1038/nbt.2726. View

20.

Gibson D, Young L, Chuang R, Venter J, Hutchison 3rd C, Smith H . Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods. 2009; 6(5):343-5. DOI: 10.1038/nmeth.1318. View