Bioengineered Guide RNA Recruits Cellular Enzymes for Editing

A new RNA editing technology could make it easier to repair disease-causing mutations in RNA without compromising precision or efficiency. Using a bioengineered guide RNA that recruits a cell’s own RNA-editing enzymes, a team at University of California San Diego, achieved 33% reduction in buildup of complex sugars in mice with Hurler syndrome.

The technology uses RNA-editing enzymes known as adenosine deaminases acting on RNA (ADARs) that naturally occur in the body’s cells. ADARs work by binding to RNA and converting some of the adenosine (A) bases to inosine (I), which is read by the cell’s translation machinery as guanosine (G). Researchers have been exploring RNA editing approaches with ADARs to correct the G-to-A mutation behind genetic disorders such as cystic fibrosis, Rett syndrome and Hurler syndrome.

A big advantage of RNA editing over DNA editing is that changes to RNA are only temporary, since RNA has a short lifespan. So if off-target edits occur, they would be short-lived.

To make a targeted A-to-I edit on RNA using ADARs, a short accessory strand of RNA—called a guide RNA—is needed to bring ADARs to the target. A big challenge with this approach is that traditional guide RNAs are not efficient at using native ADARs in the cell, so they require external ADARs be brought into the cell, according to Prashant Mali, a bioengineering professor at the UC San Diego Jacobs School of Engineering. But this can make delivery complicated and result in more off-target effects, he adds.

To overcome these issues, Mali and colleagues engineered a new kind of guide RNA—one that is extremely effective at recruiting the cell’s own ADARs to make edits at a precise target RNA region. “We can simply deliver just a small piece of RNA inside the cell and repair mutations in vivo. We don’t have to provide any extra enzymes,” said Mali.

The bioengineered guide RNAs developed by Mali and his colleagues are also longer than traditional guide RNAs, which makes them “stickier” for ADARs already present in the cell. Their circular shape also makes them more resistant to the cell’s RNA-degrading enzymes, helping them last for days and stay on the target RNA region for longer periods of time.

As a proof of concept, the team designed the guide RNAs to target the single G-to-A mutation that causes Hurler syndrome. This mutation prevents the body from producing an enzyme that is necessary for breaking down complex sugars. Buildup of these sugars causes severe tissue damage, skeletal abnormalities, cognitive impairment, and other serious health problems. Systemic injection of the guide RNAs into diseased mice resulted in correction of 7 to 17% of the mutant RNAs after two weeks, as well as a 33% decrease in the buildup of complex sugars.

The findings were published recently in Nature Biotechnology. The research is still at an early stage, but the team will now focus on improving delivery of the guide RNAs into cells. “I’m hopeful that this work opens the door even more for RNA editing as another gene therapy tool,” said Mali.