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Scientists can now silence genes without altering underlying DNA sequence

Scientists can now silence genes without altering underlying DNA sequence
A new version of the CRISPR-Cas9 gene editing technique, called CRISPRoff, causes fewer incorrect edits -- and can be reversed -- according to researchers. Photo by qimono/Pixabay

April 9 (UPI) -- The world's most powerful gene-editing tool, CRISPR-Cas9, has enabled numerous scientific discoveries. However, the tool has one major flaw: its genetic alterations are permanent.

Now, scientists have developed a tool that can precisely control gene expression without altering the underlying DNA sequence.

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The genomic tweaks forged by the new technology -- dubbed CRISPRoff and detailed Friday in the journal Cell -- can persist for several hundred generations of cell division and are fully reversible.

"The big story here is we now have a simple tool that can silence the vast majority of genes," CRISPRoff developer Jonathan Weissman said in a press release.

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"We can do this for multiple genes at the same time without any DNA damage, with great deal of homogeneity, and in a way that can be reversed. It's a great tool for controlling gene expression," said Weissman, co-author of the new study and a professor of biology at MIT.

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CRISPR-Cas9 works by excising specific DNA sequences using a gene-cutting protein, Cas9, sourced from bacterial immune systems.

Shepherded by a gene-targeting RNA, the protein snips out bits of code. When a cell's machinery automatically repairs the damage, new sequences are formed.

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Researchers have used CRISPR-Cas9 to turn genes on and off, but the alterations are permanent and not always as precise as scientists would like. Sometimes, CRISPR-Cas9 alterations yielded unintended cellular changes.

"As beautiful as CRISPR-Cas9 is, it hands off the repair to natural cellular processes, which are complex and multifaceted," Weissman said. "It's very hard to control the outcomes."

These problems inspired researchers to develop new tools for epigenetic alterations -- changes affecting gene expression, but not the DNA code itself.

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Epigenetic changes can be made by applying chemical tags to certain gene sequences. These tags prevent RNA from recognizing and transcribing the DNA code, effectively silencing expression of the genetic sequence.

Scientists have previously created tools to apply these tags, but they required target cells to continually express artificial proteins to maintain the epigenetic alteration.

"With this new CRISPRoff technology, you can [express a protein briefly] to write a program that's remembered and carried out indefinitely by the cell," said study co-author Luke Gilbert.

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"It changes the game so now you're basically writing a change that is passed down through cell divisions -- in some ways we can learn to create a version 2.0 of CRISPR-Cas9 that is safer and just as effective, and can do all these other things as well," said Gilbert, an assistant professor at the University of California San Francisco.

The new CRISPRoff system uses a tiny protein factory, guided by sequence-targeting RNA, to attach methyl groups onto specific strands of genetic code, effectively turning off a target gene sequence.

Special enzymes can be used to remove the methyl tags and reverse the alteration.

In lab tests, researchers found their system worked not only for genes themselves, but also DNA sequences that dictate gene expression.

"That was a huge shock even for us, because we thought it was only going to be applicable for a subset of genes," said first author James Nuñez, a postdoctoral researcher in Weissman's lab.

Researchers were surprised to find their new technology worked on a wide variety of genes, including genes scientists suspected would prove immune to the effects of methyl tags.

To test the technology's potential, researchers decided to silence specific genes in human stem cells, causing the cells to differentiate into neurons.

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The scientists also used CRISPRoff to silence genes controlling the production of Tau, a protein implicated in the development of Alzheimer's disease.

"What we showed is that this is a viable strategy for silencing Tau and preventing that protein from being expressed," Weissman said. "The question is, then, how do you deliver this to an adult? And would it really be enough to impact Alzheimer's? Those are big open questions, especially the latter."

Scientists hope this new ability to silence any part of the human genome will lead to powerful insights into functionality of the human genome, as well as inspire new therapies for a variety of diseases and genetic disorders.

It's possible, researchers suggest, that CRISPRoff can be used to make epigenetic alterations that persist through generations.

"I think our tool really allows us to begin to study the mechanism of heritability, especially epigenetic heritability, which is a huge question in the biomedical sciences," Nuñez said.

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