New genetic tools correct incorrect genetic bases in DNA and RNA

More targeted correction: Errors in the genetic material could be repaired more easily in the future. Researchers have developed two new repair tools that can transform a wrong gene base into the right one - without having to cut out the genetic components. Particularly exciting: one of these systems repairs such point mutations not in the DNA, but in the RNA. This results in the formation of correct proteins without the need for direct interactions with the genome.

Two new gene approaches can chemically correct pathogenic point mutations directly in DNA and RNA. © ktsimage / thinkstock

The genuine gene scissor CRISPR / Cas9 is a breakthrough, because it can be used to repair mutations in the genome more easily and more precisely than before. Researchers have already used it to cure mice from Duchenne muscular dystrophy, to correct an Alzheimer's mutation in human cells, and to repair the gene defect of Sickle-cell disease. Ethically controversial interventions into the genotype of embryos have already been tested using this method.

Code correction without cutting

However, the CRISPR gene scissor has a disadvantage: it removes faulty DNA sequences in a targeted manner, but is dependent on the repair mechanisms of the cell for the subsequent insertion of the correct code. If these cell systems do not work or only weakly, the success rate of the genetic repair is also low.

At least in the case of mutations of only single DNA bases, however, there would be an alternative: a repair system that does not cut out the defective parts but converts them chemically to the correct base directly in the DNA strand. This is mainly due to the fact that a large part of the pathogenic point mutation is due to a certain change in the base: Instead of Guanine, an Adenine sits in the DNA strand.

Adenine becomes Guanine

This mutation can now be correct by the new genetic tool from Nicole Gaudelli and her colleagues at Harvard University. It consists of a modified form of the genetic scissor CRISPR / Cas9, to which the researchers have attached another enzyme. This enzyme, which is isolated from bacteria, can convert the gene base Adenine to Inosine by chemical rearrangement - a base which is read by the cell machinery as Guanine.

We have developed a new base editor - a molecular machine that can correct G to A mutations in the genome of living cells - in a programmable, irreversible, and efficient way, says Guadellis colleague David Liu.

The fact that this genetic tool works was confirmed by tests with human cell cultures. In one of them, the researchers corrected a mutation that caused the hereditary hemochromatosis of the blood (also known as Iron overload). In a second test, they produced a sequence that protects the red blood pigment against certain blood diseases.

Correction of RNA instead of DNA

The second new gene tool is not based on the DNA, but on the RNA - the bio-molecule that copies the DNA blueprints and brings them to the protein factories of the cell. The key point: If a defective sequence is corrected in the messenger RNA, a functional protein can be generated without the need for an intervention on the DNA. Soiling mutations can be repaired without lasting and possibly ethically controversial changes of the genetic material.

David Cox and his colleagues from the Broad Institute and Harvard University have initially combined the CRISPR molecule with a Cas13 enzyme for their system called REPAIR. This ensures that the gene tool attaches to the RNA instead of the DNA. This construct was then supplemented by a protein that converts the RNA base Adenine to Inosine - similar to the ABE system of the first team.

This gene tool proved to be successful in the first tests: in human cell cultures, the REPAIR system corrected various Guanine-to-Adenine mutations, including two that cause hereditary diseases. The result: REPAIR reversed the error in 20 to 40 percent of the RNA. At the same time, only a few so-called off-target effects occurred - changes in the base sequence made in the wrong place.

A big step forward

Generating a molecular machine that is precisely the genetic change needed to cure a disease is a big step forward, Liu says.

But both gene tools still require a lot of work and testing before they can actually be used on patients.

It remains to be clarified how these molecular complexes are best transported into the cells of a patient, how safe these gene corrections are and how effectively they function in living animals and humans. Nevertheless, both teams see promising approaches in their new repair systems, in order to correct future sick-making mutations more gently and more effectively.

Source: Nature, 2017, doi: 10.1038 / nature24644, Science, 2017, doi: 10.1126 / science.aaq0180