Editing DNA Methylation in the Mammalian Genome.
Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice, demonstrating their wide utility for functional studies of epigenetic regulation.
Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
X Shawn
XS
Liu
Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
Hao
H
Wu
Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
Xiong
X
Ji
Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
Yonatan
Y
Stelzer
Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
Xuebing
X
Wu
Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA; Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, 31-007 Kraków, Poland.
Szymon
S
Czauderna
Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
Jian
J
Shu
Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
Daniel
D
Dadon
Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
Richard A
RA
Young
Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. Electronic address: jaenisch@wi.mit.edu.
Rudolf
R
Jaenisch
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D019208:Brain-Derived Neurotrophic Factor / Q000235:genetics; D000076246:CCCTC-Binding Factor; D000076987:CRISPR-Associated Protein 9; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D018899:CpG Islands; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation / Q000235:genetics*; D000090205:DNA Methyltransferase 3A; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D044127:Epigenesis, Genetic; D000072669:Gene Editing / Q000379:methods*; D016678:Genome; D051379:Mice; D017570:MyoD Protein / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D019208:Brain-Derived Neurotrophic Factor; D000076246:CCCTC-Binding Factor; C000619698:Ctcf protein, mouse; D004268:DNA-Binding Proteins; C000715535:Dnmt3a protein, mouse; D017570:MyoD Protein; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C540839:TET1 protein, mouse; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
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