Abstract
Methylation of DNA at cytosine bases is an important DNA modification underlying normal development and disease states. Despite decades of research into the biological function of DNA methylation, most of the observations so far have relied primarily on associative data between observed changes in DNA methylation states and local changes in transcriptional activity or chromatin state processes. This is primarily due to the lack of molecular tools to precisely modify DNA methylation in the genome. Recent advances in genome editing technologies have allowed repurposing the CRISPR-Cas9 system for epigenome editing by fusing the catalytically dead Cas9 (dCas9) to epigenome modifying enzymes. Moreover, methods of recruiting multiple protein domains, including the SunTag system, have increased the efficacy of epigenome editing at target sites. Here, we describe an end-to-end protocol for efficient targeted removal of DNA methylation by recruiting multiple catalytic domain of TET1 enzymes to the target sites with the dCas9-SunTag system, including sgRNA design, molecular cloning, delivery of plasmid into mammalian cells, and targeted DNA methylation analysis.
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References
Greenberg MVC, Bourc’his D (2019) The diverse roles of DNA methylation in mammalian development and disease. Nat Rev Mol Cell Biol 20:590–607
Robertson KD (2005) DNA methylation and human disease. Nat Rev Genet 6:597–610
Edwards JR, Yarychkivska O, Boulard M et al (2017) DNA methylation and DNA methyltransferases. Epigenetics Chromatin 10:23
Lister R, Pelizzola M, Dowen RH et al (2009) Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462:315–322
Lister R, Mukamel EA, Nery JR et al (2013) Global epigenomic reconfiguration during mammalian brain development. Science 341:1237905
Christman JK (2002) 5-Azacytidine and 5-aza-2′-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene 21:5483–5495
Li J-Y, Pu M-T, Hirasawa R et al (2007) Synergistic function of DNA methyltransferases Dnmt3a and Dnmt3b in the methylation of Oct4 and Nanog. Mol Cell Biol 27:8748–8759
Liao J, Karnik R, Gu H et al (2015) Targeted disruption of DNMT1, DNMT3A and DNMT3B in human embryonic stem cells. Nat Genet 47:469–478
Duymich CE, Charlet J, Yang X et al (2016) DNMT3B isoforms without catalytic activity stimulate gene body methylation as accessory proteins in somatic cells. Nat Commun 7:11453
Pflueger C, Swain T, Lister R (2019) Harnessing targeted DNA methylation and demethylation using dCas9. Essays Biochem 63:813
Lei Y, Huang Y-H, Goodell MA (2018) DNA methylation and de-methylation using hybrid site-targeting proteins. Genome Biol 19:187
Chen H, Kazemier HG, Groote ML et al (2014) Induced DNA demethylation by targeting ten-eleven translocation 2 to the human ICAM-1 promoter. Nucleic Acids Res 42:1563–1574
Maeder ML, Angstman JF, Richardson ME et al (2013) Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins. Nat Biotechnol 31:1137–1142
Grimmer MR, Stolzenburg S, Ford E et al (2014) Analysis of an artificial zinc finger epigenetic modulator: widespread binding but limited regulation. Nucleic Acids Res 42:10856–10868
Valton J, Dupuy A, Daboussi F et al (2012) Overcoming transcription activator-like effector (TALE) DNA binding domain sensitivity to cytosine methylation. J Biol Chem 287:38427–38432
Pflueger C, Tan D, Swain T et al (2018) A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs. Genome Res 28:1193–1206
Hilton IB, D’Ippolito AM, Vockley CM et al (2015) Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat Biotechnol 33:510–517
O’Geen H, Ren C, Nicolet CM et al (2017) dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res 45:9901–9916
Choudhury SR, Cui Y, Lubecka K et al (2016) CRISPR-dCas9 mediated TET1 targeting for selective DNA demethylation at BRCA1 promoter. Oncotarget 7:46545–46556
Liu XS, Wu H, Ji X et al (2016) Editing DNA methylation in the mammalian genome. Cell 167:233–247.e17
Xu X, Tao Y, Gao X et al (2016) A CRISPR-based approach for targeted DNA demethylation. Cell Discov 2:16009
Morita S, Noguchi H, Horii T et al (2016) Targeted DNA demethylation in vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat Biotechnol 34:1060–1065
Gallego-Bartolomé J, Gardiner J, Liu W et al (2018) Targeted DNA demethylation of the Arabidopsis genome using the human TET1 catalytic domain. Proc Natl Acad Sci U S A 115:E2125–E2134
Li L-C, Dahiya R (2002) MethPrimer: designing primers for methylation PCRs. Bioinformatics 18:1427–1431
Acknowledgments
TN was supported by the Forrest Research Foundation PhD Scholarship. RL was supported by a Sylvia and Charles Viertel Senior Medical Research Fellowship, NHMRC Investigator Grant (GNT1178460), and a Howard Hughes Medical Institute International Research Scholarship.
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Nguyen, T.V., Lister, R. (2021). Genomic Targeting of TET Activity for Targeted Demethylation Using CRISPR/Cas9. In: Bogdanovic, O., Vermeulen, M. (eds) TET Proteins and DNA Demethylation. Methods in Molecular Biology, vol 2272. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1294-1_10
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DOI: https://doi.org/10.1007/978-1-0716-1294-1_10
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