StemCell Therapy

Scientists have figured out how to modify the unrivaled gene-editing tool CRISPR to extend its reach to the epigenome, which controls how genes are switched on or off.

The researchers from the University of California, San Francisco, and MITs non-profit Whitehead Institute for Biomedical Research and have already used the tool in the lab to mostly deactivate the gene that makes the protein Tau, which has been implicated in Alzheimers disease.

The novel CRISPR-based tool called CRISPRoff allows scientists to switch off almost any gene in human cells without making a single edit to the genetic codeand once a gene is switched off, it remains inert in the cells descendants for hundreds of generations, unless it is switched back on with a complementary tool called CRISPRon.

Because the epigenome plays a central role in many diseases, from viral infection to cancer, CRISPRoff technology may one day lead to powerful epigenetic therapies that are safer than conventional CRISPR therapeutics because it doesnt involve any DNA edits.

Though genetic and cellular therapies are the future of medicine, there are potential safety concerns around permanently changing the genome, which is why were trying to come up with other ways to use CRISPR to treat disease, said Luke Gilbert, PhD, a professor at UCSF and co-senior author of the new paper, published in the April 9 journal Cell.

Conventional CRISPR is equipped with two pieces of molecular hardware that make it an effective gene-editing tool. One component is a DNA-snipping enzyme, which gives CRISPR the ability to alter DNA sequences. The other is a homing device that can be programmed to zero in on any DNA sequence of interest, imparting precise control over where edits are made.

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To build CRISPRoff, the researchers dispensed with conventional CRISPRs DNA-snipping enzyme function, but retained the homing device, creating a stripped-down CRISPR capable of targeting any gene. Then they tethered an enzyme to this barebones CRISPR. But rather than splicing DNA, this enzyme acts on the epigenome, which consists of proteins and small molecules that latch onto DNA and control when and where genes are switched on or off.

The new tool targets a particular epigenetic feature known as DNA methylation, which is one of many molecular parts of the epigenome. When DNA is methylated, a small chemical tag known as a methyl group is affixed to DNA, which silences nearby genes. Although DNA methylation occurs naturally in all mammalian cells, CRISPRoff offers scientists unprecedented control over this process.

Another tool described in the paper, called CRISPRon, removes methylation marks deposited by CRISPRoff, making the process fully reversible.

Now we have a simple tool that can silence the vast majority of genes, said Jonathan Weissman, PhD, Whitehead Institute member, co-senior author of the new paper and a former UCSF faculty member. We can do this for multiple genes at the same time without any DNA damage, and in a way that can be reversed. Its a great tool for controlling gene expression.

Based on previous work by a group in Italy, the researchers were confident that CRISPRoff would be able to silence specific genes, but they suspected that some 30 percent of human genes would be unresponsive to the new tool.

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DNA consists of four genetic letters A, C, G, T but, in general, only Cs next to Gs can be methylated. To complicate matters, scientists have long believed that methylation could only silence genes at sites in the genome where CG sequences are highly concentrated, regions known as CpG islands.

Since nearly a third of human genes lack CpG islands, the researchers assumed methylation wouldnt switch these genes off. But their CRISPRoff experiments upended this epigenetic dogma.

What was thought before this work was that the 30 percent of genes that do not have CpG islands were not controlled by DNA methylation, said Gilbert. But our work clearly shows that you dont require a CpG island to turn genes off by methylation. That, to me, was a major surprise.

Easy-to-use epigenetic editors like CRISPRoff have tremendous therapeutic potential, in large part because, like the genome, the epigenome can be inherited.

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When CRISPRoff silences a gene, not only does the gene remain off in the treated cell, it also stays off in the descendants of the cell as it divides, for as many as 450 generations.

To the researchers surprise, this held true even in maturing stem cells. Though the transition from stem cell to differentiated adult cell involves a significant rewiring of the epigenome, the methylation marks deposited by CRISPRoff were faithfully inherited in 90 percent of cells that made this transition, which showed that cells retain a memory of epigenetic modifications made by the CRISPRoff system even as they change cell type.

They selected one gene to use as an example of how CRISPRoff might be applied to therapeutics: the gene that codes for Tau protein, which is implicated in Alzheimers disease. After testing the method in neurons, they discovered that using CRISPRoff could be used to turn Tau expression downalthough not entirely off. What we showed is that this is a viable strategy for silencing Tau and preventing that protein from being expressed, says Weissman. The question is, then, how do you deliver this to an adult? And would it really be enough to impact Alzheimers? Those are big open questions, especially the latter.

Even if CRISPRoff does not lead to Alzheimers therapies, there are many other conditions it could potentially be applied to. Their findings suggest that CRISPRoff would only need to be administered once to have lasting therapeutic effects, making it a promising approach for treating rare genetic disorders including Marfan syndrome, which affects connective tissue, Jobs syndrome, an immune system disorder, and certain forms of cancer that are caused by the activity of a single damaged copy of a gene.

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While delivery to specific tissues remains a challenge, we showed that you can deliver it transiently as a DNA or as an RNA, the same technology thats the basis of the Moderna and BioNTech coronavirus vaccine, Weissman says.

Because the epigenome plays a central role in so many diseases, this exciting new technology may one day lead to powerful therapies to tackle our deadliest foes, although further work is needed to realize its full therapeutic potential.

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