Scientists use CRISPR/Cas9 to flip DNA methylation states in vivo

CAMBRIDGE, Mass.  – Whitehead Institute scientists have deciphered how to use a modified CRISPR/Cas9 gene editing system to change genes’ methylation state, thereby activating or silencing those genes. Proper methylation is critical for normal cellular operations and altered methylation has been linked to many diseases, including neurological disorders and cancer.

“I think this is an important tool for looking at the epigenetic state—the methylation state—of the regulatory sequences that control gene expression,” says Whitehead Founding Member Rudolf Jaenisch, who is also a professor of biology at MIT. “Epigenetics is very important for disease states. Therefore, being able to change a gene or a regulatory sequence’s methylation state in vivo is significant.”

Although all cells in an organism contain the same recipes for proteins, the cell’s type determines which recipes, or genes, are used. One mode for controlling gene expression is methylation—the addition or removal of molecules called methyl groups. When methyl groups are attached to DNA, that segment is usually turned off, whereas DNA without methyl groups is generally active and available for transcription. Proper methylation is critical during development and for normal cellular operations; incorrect methylation can cause disease, including fragile X syndrome and cancer.

To target and alter the methylation of specific genes, researchers in the Jaenisch lab used a modified form of the CRISPR/Cas9 genome editing system in which a catalytically inactive Cas9 (dCas9) is fused with enzymes that add (Dnmt3a) or remove methyl groups (Tet1). Their work is described this week in the journal Cell.

After a successful test run in mouse cells, the team put the system through its paces. They demethylated regulatory sequences of a key gene in mouse neurons that is usually turned on by neuronal activity, and facilitated the conversion of mouse skin cells to muscle cells by demethylating the enhancer of a master regulator for myogenesis called MyoD.

Changing DNA’s methylation state can also alter the greater three-dimensional architecture of DNA, called CTCF-mediated loops, which are essential for the proper expression of many critical genes. These loops act as “insulated neighborhoods” that bring genes and their super-enhancers in close enough proximity for the super-enhancers to promote the genes’ expression. By methylating the spot on the DNA where CTCF proteins bind to form a loop, the team can disrupt the loop’s construction and alter the target genes’ expression.

In the future, changing genes’ methylation state could be used to treat diseases caused by abnormal methylation. To test the system’s capabilities in vivo, the team used it to activate a gene in the hippocampal neurons of transgenic mice by demethylating the gene’s promoter.

“This system is very versatile,” says Shawn Liu, a postdoctoral researcher in the Jaenisch lab and co-author of the Cell paper. “It can be used to demethylate or methylate many targeted regions in the mammalian genome so we can study their biological or pathological significances.”

Co-author Hao Wu shares Liu’s enthusiasm.

“We think that this system might be a game changer—to study the DNA methylation in the genome and also to control gene expression in a targeted fashion,” says Wu, who is a postdoctoral researcher in the Jaenisch lab. “And we think that this might be a good tool for therapeutic approaches in the future.”

This work was supported by the National Institutes of Health (NIH grants HD045022 and HG002668), Damon Runyon Cancer Foundation, the Brain and Behavior Research Foundation, Helen Hay Whitney Foundation, National Science Centre in Poland (HARMONIA grant No. UMO-2014/14/M/NZ1/00010), and the Human Frontier Science Program. Rudolf Jaenisch is co-founder of Fate Therapeutics and Fulcrum Therapeutics and an adviser to Stemgent, and Richard Young is a founder of Syros Pharmaceuticals.

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Rudolf Jaenisch's primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also a professor of biology at Massachusetts Institute of Technology.

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Full Citation:

 X Shawn Liu  et al. “Editing DNA methylation in the mammalian genome.” Cell, September 22, 2016.