CRISPR-Cas9 gene editing has proved useful in blocking specific genes to correct mutations, but it makes permanent changes to the genome. A team from the Broad Institute has devised a CRISPR-based system that targets RNA in a way that makes reversible changes to DNA possible.
The system, dubbed REPAIR (RNA Editing for Programmable A to I Replacement) can edit single nucleosides, or the “letters” that make up the RNA helix. Specifically, it can change the nucleoside adenosine (A) to inosine (I), which is read as guanosine (G) inside cells. This could become a treatment for diseases in which a G-to-A mutation plays a role, including Duchenne muscular dystrophy and Parkinson’s disease.
CRISPR-Cas9 employs Cas9 enzymes, which target and cut DNA. The REPAIR system is based on Cas13, an enzyme that does the same, but for RNA. The scientists created a deactivated version of the Cas13 enzyme, PspCas13b, which binds precisely to a stretch of RNA, but does not cut it. They combined the enzyme with the protein ADAR2, which switches A to I in RNA transcripts, and then improved the tool to cut down on off-target effects.
"So far, we've gotten very good at inactivating genes, but actually recovering lost protein function is much more challenging. This new ability to edit RNA opens up more potential opportunities to recover that function and treat many diseases, in almost any kind of cell,” said Feng Zhang, core institute member at the Broad Institute, in a press release.
The team tested their tool against RNA mutations that cause Fanconi anemia and X-linked nephrogenic diabetes insipidus, finding that the system repaired the mutations.
"REPAIR can fix mutations without tampering with the genome, and because RNA naturally degrades, it's a potentially reversible fix," said co-first author David Cox, a graduate student in Zhang's lab. The findings appear in Nature.
Developing CRISPR-based methods that go beyond cutting DNA is not new. University of California, San Francisco scientists “blunted” CRISPR so it could target the 98% of the human genome that does not directly code for proteins. Instead, this “gentler” CRISPR targets promoters and enhancers—gene sequences that switch other genes on—and activates them to see how they affect gene expression.
A team from UC San Diego is also working to correct mistakes in RNA to treat a group of diseases that are caused by too many repeats in RNA sequences, causing the RNA to clump together in cells. The RNA-targeted CRISPR managed to clear the majority of clumps and errant RNA sequences in models of Huntington’s and a type of ALS.
The Broad researchers plan to make a more efficient version of their system, REPAIRv2, and create a delivery system to deploy it in animal models. They also plan to explore using the system to make other nucleoside conversions.