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Researchers devise a novel, high-precision, gene-editing tool to fix genetic diseases

18/11/19 - Gene editing, Gene Therapy,

In recent years, a powerful gene-editing technology called CRISPR-Cas9 has revolutionized biology turning the process of genome modification into an easy and inexpensive process. This revolutionary technique makes use of molecular tools that allow precise identification of DNA fragments –for example a gene– and either introduce modifications, delete a genomic fragment, or even insert a new DNA fragment or a whole gene in the same location.

Gene editing is being applied in many laboratories both in cultured cells and in model organisms, to study the molecular basis of certain diseases and to develop future therapies to treat them. In the field of inherited eye diseases, the American company Editas Medicine has developed the first in vivo CRISPR-Cas9 gene-editing therapy for Leber congenital amaurosis (LCA). The therapy is addressed at LCA patients who harbour a particular mutation in the CEP290 gene and, Editas Medicine, together with Allergan, have just started (September 2019) a clinical trial with 18 patients to evaluate the safe and efficacy of this treatment.

Despite being a very powerful tool, the CRISPR-Cas9 gene editing approach is not always accurate as it can produce fortuitous changes, something that has been used to suppress the function of a gene and study its effects. However, when the aim is to correct a specific mutation causing a monogenic disease, the inaccuracy of the current technique has to be avoided.

That is why a team at the Broad Institute of Harvard University and the Massachusetts Institute of Technology, led by David R. Liu, has developed a new gene-editing method, named “prime editing”, that is much more precise. In a recent article in Nature, researchers claim that 89% of the disease-associated mutations can be corrected using this new method. Researchers have shown the high level of efficiency of the prime-editing method in human cells in culture to correct the genetic defects responsible for sickle cell anemia and Tay-Sachs disease.

How does prime editing work?

Like the conventional CRISPR, prime editing uses an RNA molecule to guide and locate the genome fragment to be corrected coupled to the Cas9 protein, a molecular scissor of DNA. The prime editing Cas9, unlike the initial method, generates a nick in only one strand of DNA and not both. The RNA guide is now longer and comprises a target site DNA-binding region –preparing the DNA for modification– and an adjacent sequence fragment to be used as a template. The Cas9 protein is also different: scientists have combined it with another protein called reverse transcriptase (RT), which copies genetic information from RNA to DNA. Starting at the point of the nick, the RT synthesises a new fragment of DNA using the RNA guide as a template.

The new technique also uses the cell’s own machinery. In this case, a protein called endonuclease will remove the DNA fragment that harbours the mutation and and makes the proper ligation with the new corrected fragment. At this point, the mutation has been corrected in only one of the two DNA strands, generating a mismatch. Then, a different guide RNA comes into play that directs the prime-editing complex (Cas9-RT) towards the unedited strand to nick it, after which the cellular machinery will replace it and fill the gap, using the corrected strand as a template. Prime editing is more efficient and less prone to error making than the conventional CRISPR. Depending on the cell type, ranges between 20 to 50 percent, and could reach 78 percent.

At the moment, this new technique has been tested in cultured cells, but surely we will soon begin to see its applications in model organisms and in humans. The eye, with its relative ease of access to the cells in which the therapy needs to be delivered, will undoubtedly be one of the first tissues in which the “prime editing” technology will be applied.

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