CRISPR gene-editing tool targets RNA, tackles dementia

Scientists at the Salk Institute have used CRISPR gene-editing technology to target RNA — as opposed to DNA — and in the process corrected a protein imbalance in cells from a dementia patient.

Called CasRx, the tool opens up the vast potential of RNA and proteins to genetic engineering, giving researchers a powerful way to develop new gene therapies as well as investigate fundamental biological functions. It has been described in the journal Cell.

Based on the immune system of bacteria, CRISPR-Cas9 gene editing technology can be described as the use of targeted ‘molecular scissors’ to cut and replace disease-causing genes with healthy ones. The cutting is performed by the Cas9 protein, while other parts of the system act as guides that instruct where Cas9 should cut the DNA.

Scientists have been harnessing Cas9 molecular scissors for a few years now in combination with artificial guides to modify genes in bacteria, plants and animals. But while these scissors typically cut DNA, the Salk team decided to search bacterial genomes for new CRISPR enzymes that could instead target RNA — a working copy of DNA that is translated into proteins. As faulty RNA is the cause of many genetic diseases, the team’s hope was that they could engineer CRISPR enzymes to address problems with RNA and the resulting proteins.

“The DNA in your cells are largely the same, whereas the RNA product is really what’s changing, and mediates dynamic processes like inflammation or behaviour,” said Patrick Hsu, senior author of the study. “And in fact, many genetic diseases are actually caused by defects directly at the RNA level. And so by targeting RNA we can start to try to manipulate these processes.”

For example, a given RNA message can be expressed at varying levels and its balance relative to other RNAs is critical for healthy function. Furthermore, RNA can be ‘spliced’ in various ways to make different proteins, but problems with splicing can lead to diseases such as spinal muscular atrophy, atypical cystic fibrosis and frontotemporal dementia (FTD).

“What splicing does is, it basically modifies the RNA message to turn into two different kinds of proteins, for example,” said Salk Research Associate Silvana Konermann, the paper’s first author. “And this balance of these two different types of proteins is dysregulated in certain diseases, like neurodegeneration.”

So how did the researchers manage to target RNA? As explained by Konermann, “We began the project with the hypothesis that different CRISPR systems may have been specialised throughout an evolutionary arms race between bacteria and their viruses, potentially giving them the ability to target viral RNA.”

The team developed a computational program to search bacterial DNA databases for the telltale signatures of CRISPR systems: patterns of particular repeating DNA sequences. In doing so, they discovered a family of CRISPR enzymes that targets RNA, and called it Cas13d.

The team realised that, just like the Cas9 family, Cas13d enzymes originating from different bacterial species would vary in their activity, so they ran a screen to identify the best version for use in human cells. That version turned out to be from the gut bacterium Ruminococcus flavefaciens XPD3002, which led them to name their tool CasRx.

“Once we engineered CasRx to work well in human cells, we really wanted to put it through its paces,” said Konermann. Deciding to focus on a disease-related condition, the researchers engineered CasRx to tackle FTD — a type of dementia in which the ratio of two versions of the Tau protein (also implicated in Alzheimer’s disease) is out of balance in neurons.

“The Tau transcript can be translated into two different types of Tau protein,” explained Konermann. “And in a healthy cell, there’s a very finely controlled balance between those two different types of Tau proteins. In the diseased cell, one type of protein now is dominating, and more than it should be.”

The team genetically engineered CasRx to target RNA sequences for the version of the Tau protein that is overabundant. They did this by packaging CasRx into a virus and delivering it to neurons grown from an FTD patient’s stem cells.

According to the researchers, CasRx was 80% effective in rebalancing the levels of Tau protein to healthy levels. Konermann stated, “By using our RNA-targeting CRISPR protein to target specific elements inside the RNA message, we’re able to reset the balance between these two kinds of Tau proteins.”

The Salk team is not the first to develop molecular scissors to target RNA. Earlier this month, German scientists showed that the Cas9 protein of the foodborne pathogen Campylobacter jejuni is capable of cutting RNA — a revelation that came soon after two other research groups reported similar findings with Cas9s from two other bacteria.

The Salk team does, however, believe CasRx has certain advantages compared to other RNA-targeting technologies, citing its small size (making it easier to package into therapeutically relevant viral vectors), its high degree of effectiveness and the fact that it created no discernible off-target effects compared to RNA interference. The researchers are excited about the possibilities their tool opens up for exploring new biological questions about RNA and protein function, as well as therapies to tackle RNA and protein-based diseases.

“I think we’re really only scratching the surface of what we can do with these genetic engineering tools,” said Hsu. “And by targeting RNA, the hope is we can develop new types of intelligent therapies that can respond to the state of a cell, and not just the genome that encodes it.”

Image caption: CasRx (magenta) targeting RNA in the nucleus of human cells (grey). Image credit: Salk Institute.