Specificity and efficiency in targeting is one of the hallmark aims of any CRISPR system. The ability to edit a genome is useless without the capability to target a particular sequence and reliably alter it. Thus, work has been perpetually ongoing in the hunt to find better CRISPR systems or to even design one from scratch. Though, at the same time, there has also been a search for ways to use CRISPR that would lessen the public concern about gene editing in humans.

Subverting Expectations

Researchers at the Broad Institute, MIT, and specifically the Feng Zhang lab, all of which were early adopters of CRISPR technology, have unveiled a new solution to both problems. If there are worries about alteration of DNA, then how about RNA instead? The impact on the body will still allow for disease treatment and other changes, while not being a permanent adjustment in the makeup of the genome. If necessary, the change can be reversed by removing the CRISPR machinery.

The new tool they have created is the result of an arduous scan through available bacteria to find one with the most efficient CRISPR proteins of the type they desired. Specifically, this meant the Cas13 family, of which C2c2 was formerly included and was later renamed to Cas13a. This group of Cas proteins uses a complex that focuses on cutting single stranded RNA rather than double stranded DNA.

They ultimately settled on an additional editor enzyme titled PspCas13b, which originates from the bacteria species Prevotella. It had the greatest success at inactivating RNA, a necessary part of the Cas13 system they were creating. With this, they were able to conceive a device they termed REPAIR, RNA Editing for Programmable A to I Replacement.

Correcting Mutations

As the name suggests, this CRISPR tool is able to turn A RNA nucleotides into I (inosine) ones. The latter are read as G nucleotides by the cellular translational machinery. This specialized alteration is very particular, in that it only affects one kind of nucleotide change, but it is also highly targeted thanks to that. Which is necessary when the target will be all the RNA transcripts of a specific gene sequence being created by a cell.

The scientists were looking for this singular kind of change first because many degenerative disorders, such as Duchenne muscular dystrophy and Parkinson’s are the result of a G to A mutation in a gene. Now, this tool can be used to alter the RNA transcripts made from these faulty genes and convert them to the correct sequences before they are translated into proteins. In essence, having the body create the correct proteins for biological function even while the genes in question remain mutated and inaccurate.

Its specific interaction uses a version of the PspCas13b enzyme that has been “blunted” so that it can no longer cut RNA and instead binds to the target section. Additionally, the enzyme has been fused to another enzyme called ADAR2, a member of the adenosine deaminase family that, as one might expect, converts A nucleotides to I. So the first enzyme is used to make contact with the RNA at the desired position and the fused ADAR2 enacts the change. Of course, with the CRISPR Cas13 system being used to guide the fused enzymes to the RNA in the first place.

Efficiency and Expansion

The original 1.0 version of REPAIR the researchers created still had too high of an off-target edit rate for their liking, so further experimentation resulted in REPAIR version 2 that reduced the number of off-target edits in the entirety of a cell’s RNA transcripts from 18,385 to 20. At the same time, they were able to increase the successful A to I change rate in the whole of the transcribed target RNA from 20% to 51%, more than enough to alleviate the aforementioned maladies. They also believe that further enhancement to REPAIR’s efficiency is possible.

To test its effects, the researchers made synthetic versions of genes that cause two well known disorders and introduced them into human cell cultures so they would begin expressing the genes. With REPAIR then added to the cells, they were able to confirm its alteration of the RNA transcripts from said genes.

The next step they want to focus on, in addition to efficiency increases, is to formulate a delivery system capable of inserting REPAIR into cells from a variety of animal models, opening up the kinds of research the tool can be used for. Other supplementary tools able to make other kinds of nucleotide changes other than A to I are in the works as well.

Targeted Success

All the universities, organizations, and people involved have furthermore reconfirmed their commitment to open source research and have pledged to share REPAIR around the world with other scientists so even greater amounts of experimentation can be done. Thus far, the REPAIR tool has been given to over 2,200 laboratories in 61 countries.

With amazing new foundational mechanisms like this coming out of contemporary science and being shared with everyone, the medical and biological fields of science continue to accelerate their progress in making this world a better place for everyone.

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Photo CCs: Human Cell from Wikimedia Commons