Neat scissors
There are high hopes for the therapeutic potential of the CRISPR-Cas9 genome editing tool. These "genetic scissors" can be used to excise and repair mutated genes responsible for hereditary diseases. But, despite the accuracy with which the tool is able to determine its target in the genome, sometimes incisions are made in areas that are very similar to the target sequence, but located in completely different areas of DNA. These off-target mutations can have unexpected consequences. And even if CRISPR-Cas9 makes an incision in the right place, errors may occur during the restoration of DNA strands – the so-called non-target mutations.
Distance between sections
Errors arise mainly due to the fact that in the classic version of CRISPR-Cas9, both DNA strands are cut at once. Researchers from the Helmholtz Association's Max Delbrück Center for Molecular Medicine in Berlin and Humboldt University in Berlin have presented a new improved approach – the spacer-nick separation method. It uses a modified pair of molecular scissors – the enzyme nicase, which makes incisions at two different points on opposite strands of DNA. This ensures that the two incisions are located at a distance of 200 to 350 base pairs from each other and will help avoid double-stranded breaks in the DNA. Experiments with hematopoietic stem cells and T-lymphocytes have shown that this is the optimal distance to minimize non-target mutations.
Efficient and error-free
Scientists have quantitatively compared the effectiveness of genome editors. Using the classical CRISPR-Cas9 method, non-target mutations were found in more than 40% of interventions, the spacer-nick system reduced this figure to 2%. The number of non-target mutations is much more difficult to determine, but the researchers noted that they occurred relatively often when using classical genetic scissors, and when editing spacer-nick were rare or absent altogether.
The exact mechanism by which genetic material is restored after incisions with the help of nicase remains unclear.
From the point of view of efficiency, spacer-nick is not inferior to conventional CRISPR-Cas9: both methods can successfully restore 20-50% of the treated cells. This is probably enough to cure patients with a hereditary disease that is associated with only one altered gene, for example beta-thalassemia, which is associated with improper hemoglobin synthesis, or severe congenital neutropenia, which is characterized by a significantly reduced number of granulocytes.
The authors hope that other researchers will pick up their idea and test spacer-nick first on animal models, and soon on the first human patients. The principle underlying the therapy is simple: hematopoietic stem cells are taken from people with a monogenic hereditary disease, then spacer-nick restores defective genes directly in cell culture. Once the genetic scissors have done their job, the restored stem cells are injected back into the patient, where they produce new and, most importantly, healthy blood cells.
Article N.Tung Tran et al. Precise CRISPR-Cas–mediated gene repair with minimal off-target and unintended on-target mutations in human hematopoietic stem cells is published in the journal Science Advances.
Aminat Adzhieva, portal "Eternal Youth" http://vechnayamolodost.ru based on EurekAlert: A fine-tuned gene editor.