The CRISPR-Cas9 gene editing tool shows incredible promise in treating a wide range of diseases, from HIV to cancer. But the technology isn't without controversy, as the long term effects of cutting DNA in living organisms isn't fully known. Now, scientists from the Salk Institute have modified CRISPR to work without making any cuts, switching targeted genes on and off instead, and demonstrated its effectiveness by treating diabetes, muscular dystrophy and other diseases in mice.

The CRISPR gene-editing tool is one of the most important scientific breakthroughs in years, with the potential to reverse the effects of disease or even snip them out of the genome at the embryo stage. But as exciting as it is, a recent study found that the cut-and-paste method may introduce unintentional mutations into the genome, and although this study was later contested, safety remains a concern at this early stage in the technology.

"Although many studies have demonstrated that CRISPR-Cas9 can be applied as a powerful tool for gene therapy, there are growing concerns regarding unwanted mutations generated by the double-strand breaks through this technology," says Juan Carlos Izpisua Belmonte, senior author of the study. "We were able to get around that concern."

Salk scientists modify CRISPR to epigenetically treat diabetes, kidney disease, muscular dystrophy

The Salk scientists adapted the regular CRISPR mechanism to influence gene activation without actually changing the DNA itself. The Cas9 enzyme normally does the cutting, so the team used a dead form of it called dCas9 that can still target genes but doesn't damage them. The active ingredients this time are transcriptional activation domains, 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.

The technique enhanced the skeletal muscle mass (top) of treated mice (right) against a control (left), as well as the growth of muscle fibers (bottom) Salk Institute

To test how well the new technique worked, the researchers experimented with mice that had three different diseases – kidney damage, type 1 diabetes and muscular dystrophy. In each case, the mice were treated with specialized CRISPR systems to increase the expression of certain genes, which would hopefully reverse the symptoms.

In the kidney-damaged mice, the team targeted two genes that play a role in kidney function. Sure enough, there was an increase in the levels of a protein linked to those genes, and kidney function improved. In the diabetic mice, the targeted genes were those that promote the growth of insulin-producing cells, and after treatment, the mice were found to have lower blood glucose levels. And finally, the treatment also worked to reverse the symptoms of muscular dystrophy.

After that promising start, further work is underway on the system. The researchers plan to try to apply the technique to other cell types to help treat other diseases, and conduct more safety tests before human trials can begin.

The research was published in the journal Cell.

Source: Salk Institute