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No-Cut, Epigenetic CRISPR Mends Defective DNA

We are at a golden age of genetic engineering, where huge advances in gene-editing technology are making it possible for scientists to tweak the DNA of different organisms with incredible, unprecedented precision.

Until just a few years ago, altering individual genes in everything from plant cells to mouse cells to human cells was a crude, laborious, and often futile process.

And now here we are, as scientists use techs like CRISPR, which harnesses the immune system of bacteria to snip individual genes, either knocking them out or even inserting new ones in their place. But all good things are well… not all good- and in case of this gene editing tool, sure enough it snips DNA at precise locations, but it is also is capable of “turning up the volume” on some genes.

This potentially overcomes the problem of the wrong genes being modified by mistake, so-called off-target effects, which is viewed as a major safety barrier to using Crispr in a clinical context.

Cutting DNA opens the door to introducing new mutations,” said Juan Carlos Izpisua Belmonte, who led the latest work at the Salk Institute in La Jolla, California. “That is something that is going to stay with us with Crispr or any other tool we develop that cuts DNA. It is a major bottleneck in the field of genetics – the possibility that the cell, after the DNA is cut, may introduce harmful mistakes.

With that in mind, a team of researchers set out to develop a take on CRISPR-Cas9 technology that wouldn’t require the DNA to be cut at all. A trial of their technique was successfully used on mice to treat several diseases. The study proves, for the first time, that the phenotype of an animal can be altered via epigenetic editing while ensuring the integrity of the DNA is preserved and no mutations are introduced.

Think of it as CRISPR disarmed. The function that guides the CRISPR system to a precise location in a genome is still there—it’s just missing the scissors. Instead, molecular switches are used to turn specific genes on and off. The resulting changes aren’t genetic—they’re epigenetic. Taken literally, epigenetic means “above the gene.”

The epigenome tells a genome what to do. By modifying the epigenome, it’s possible to control how a gene behaves without actually modifying any DNA directly. It’s sort of like gene editing, without actually doing any editing.

To accomplish this, the Salk scientists relied on what’s known as a “dead” form of Cas9. In the CRISPR system, the Cas9 enzyme is what actually does the cutting. But its dead version doesn’t cut.

The active ingredients this time were transcriptional activation domains in its place, which act like molecular switches to turn specific genes on or off. These are coupled to the dCas9, along with the usual guide RNAs that help them locate the desired section of DNA.

There’s just one problem with this technique: normally the CRISPR system is loaded into a harmless virus called an adeno-associated virus (AAV), which carries the tool to the target. But the entire protein, consisting of dCas9, the switches and the guide RNAs, is too big to fit inside one of these AAVs.

To work around that issue, the researchers split the protein into two, loading dCas9 into one virus and the switches and guide RNAs into another. The guide RNAs were tweaked to make sure both parts still ended up at the target together, and to make sure the gene was strongly activated.

No-Cut, Epigenetic CRISPR Mends Defective DNA
The team from left: Hsin-Kai (Ken) Liao, Juan Carlos Izpisua Belmonte and Fumiyuki Hatanaka

In tests using a mouse model of acute kidney disease, the technique was seen to activate damaged or silenced genes, allowing the kidney to function normally. It proved useful for helping liver cells regain the ability to produce insulin, which contributed to a mouse’s recovery from type 1 diabetes.

The team also demonstrated the capacity to recover muscle growth and function in mouse models of muscular dystrophy, a condition that’s linked to a gene mutation. But instead of seeking to correct it, the expression of genes in the same pathway was actually increased — which then took precedence over the mutation.

We are not fixing the gene; the mutation is still there,” said Belmonte, “Instead, we are working on the epigenome and the mice recover the expression of other genes in the same pathway. That is enough to recover the muscle function of these mutant mice.

No-Cut, Epigenetic CRISPR Mends Defective DNA
The Belmonte lab’s advanced in vivo Cas9-based epigenetic gene activation system enhances skeletal muscle mass (top) and fiber size growth (bottom) in a treated mouse (right) compared with an independent control (left). The fluorescent microscopy images at bottom show purple staining of the laminin glycoprotein in tibialis anterior muscle fibers.

We were very excited when we saw the results in mice,” adds Fumiyuki Hatanaka, a research associate in the lab and co–first author of the paper. “We can induce gene activation and at the same time see physiological changes.

There are still multiple steps that need to be examined before applying this method in human patients,” co-first author Hsin-Kai Liao said. “For example, it must be determined whether host immune responses against the AAV-CRISPR/Cas9 Target Gene Activation system arise in mice or large animals. Safety and ethical considerations will also have to be addressed before bringing this technique to human patients.

Belmonte and his colleagues are working to expand the new system’s application to different cell and organ types, diseases, and age-related conditions. “Our goal will be to re-activate genes silenced by ageing, or to use the system to replenish stores of adult stem cells, which promote regeneration but are typically depleted with age,” he said.

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