MIT researchers have developed REPAIR, a new version of the CRISPR/Cas9 gene editing system that can edit RNA instead of DNA — so as not to alter a person’s genome — and may one day be used to treat Parkinson’s and a variety of other diseases.
The classic CRISPR/Cas9 system involves a guide RNA (gRNA) and a protein called the Cas9 nuclease. The gRNA guides the Cas9 nuclease to a precise location in the genome, where Cas9 can cause a double stranded break. This prompts the cell’s repair machinery to fix the break, leading to mutations in the gene so as to cause it to not be expressed.
Researchers at MIT engineered a new system for mammalian gene editing, which targets the RNA sequence instead of the DNA sequence. DNA codes for RNA molecules, which then code for proteins. In this way, targeting RNA can still change the gene product (a protein), but without making a change in the entire genome.
REPAIR — the RNA Editing for Programmable A to I Replacement — can change a single RNA nucleotide, potentially reversing some of the disease-causing mutations at the RNA level.
“The ability to correct disease-causing mutations is one of the primary goals of genome editing,” Dr. Feng Zhang, the study’s senior author and an associate professor in Brain and Cognitive Sciences and Biological Engineering departments at MIT, said in a press release. “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.”
Much like DNA, RNA is made up of a sequence of letters, or nucleosides. In many human diseases, including Parkinson’s, there is often a mutation from the nucleoside G to A, and this single nucleoside change can lead to disease symptoms. REPAIR has the ability to recognize a specific A, and turn into a nucleoside called I (inosine). The I is read by cellular machinery as a G, which essentially turns back the A mutation to a G correction.
Unlike the Cas9 protein, the Cas13 enzyme targets and cuts RNA. Dr. Zhang’s research group discovered a Cas13 enzyme from the bacterial species Prevotella, called PspCas13b, which was found to be very effective at targeting RNA.
They inactivated the cleavage activity of Cas13, so it would no longer cause a double-stranded break. Instead, they fused Cas13 to a protein called ADAR2, which changes the letter A to the letter I. This leads to a simple mechanism where Cas13 seeks out the target sequence of RNA, and ADAR2 makes the conversion from A to I, leading to the formation of the correct protein.
To further improve the REPAIR system’s efficiency, the researchers also created an upgraded version, called REPAIRv2, which succeeded at the targeted edit up to 51% of the time.
To demonstrate that REPAIR can work in a disease context, researchers treated human cells with single nucleoside mutations that cause Fanconi anemia and X-linked nephrogenic diabetes insipidus. Work showed that REPAIRv2 successfully edited the mutation at the RNA level.
“The success we had engineering this system is encouraging, and there are clear signs REPAIRv2 can be evolved even further for more robust activity while still maintaining specificity,” said Omar Abudayyeh, co-first author and a graduate student in Zhang’s lab.
The team plans to continue to improve REPAIRv2 by manipulating the delivery system to improve its effectiveness when introduced into human cells.