Genome editing is a powerful technique that has the potential to correct human diseases caused by genetic errors. Many human diseases have been linked to an error, or mutation, in a gene’s DNA sequence. A common type of mutation is a single DNA nucleotide change, called a point mutation. Depending on where it’s located, a point mutation can cause a protein to work incorrectly or not at all.
Scientists recently discovered a promising DNA editing system called CRISPR/Cas9. CRIPSRs (clustered regularly interspaced short palindromic repeats) are DNA sequences that can be used to guide a DNA-cutting enzyme called Cas9 to specific places on the genome. This system can then make sequence-specific edits in the DNA. However, in many cell types, DNA repair techniques are limited in their ability to repair point mutations.
Scientists recently found that a related CRISPR system uses an enzyme called Cas13 that recognizes and cuts RNA rather than DNA. Among other functions, RNA serves as the messenger that carries instructions between DNA and the cellular machinery to make proteins. The ability to edit RNA before it’s translated into a protein could open up new therapeutic options for human diseases.
To test whether Cas13 can be used to fix point mutations in RNA, a team led by Dr. Feng Zhang at the Broad Institute of Massachusetts Institute of Technology and Harvard University carried out genome editing studies in mammalian cell lines. The research was supported by several NIH components (see full list below in the Funding section). Results were published online in Science on October 25, 2017.
The team first tested different versions of Cas13 to find which one knocks down RNA activity most robustly. They then modified the Cas13 so that it would still target RNA, but not cut it. Using this modified version, they created a targeted RNA editing system by fusing it to part of another enzyme called ADAR2. ADARs can change the nucleotide adenosine (A) to inosine (I). Because of the structural similarities between I and guanosine (G), RNAs containing an I behave as though there were a G in its place.
The team tested this RNA Editing for Programmable A to I Replacement (REPAIR) system in cells that contained gene mutations from 36 diseases. Each disease has a known point mutation at a specific site in which an A is located where a G should be. The REPAIR system was able to edit 35 of these sites, with up to 35% editing efficiency.
Using structure-guided protein engineering of ADAR2, the researchers were able to reduce the off-target effects and create the REPAIRv2 system, which also had improved specificity.
“The ability to correct disease-causing mutations is one of the primary goals of genome editing,” Zhang says. “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.”