CRISPR-Cas9: even more precise
Gene therapy is a promising strategy for the treatment of diseases caused by genetic abnormalities. CRISPR-Cas9 is becoming increasingly popular – a relatively new method of genome editing that works on the principle of scissors that cut off defective sections of DNA. The potential of CRISPR-Cas9 is difficult to overestimate, however, and it carries a number of undesirable effects due to errors in the identification of genes that need to be cut off.
A group of scientists from Osaka University (Japan), led by Shinichiro Nakada, reported on a development that modifies CRISPR-Cas9 and significantly reduces the risk of errors in the editing process.
Left: Cas9 cuts both strands of DNA, which are then restored in the presence of a donor template. On the right: the SNGD technique, in which the nucleotides of the chain with the target gene and donor DNA are partially cut out.
The CRISPR-Cas9 method consists of two components: the Cas9 protein, which acts as scissors and cuts off the defective gene, and the guide RNA, which tells the Cas9 protein which part of the DNA needs to be removed. Together, these two components can remove any gene. The problem is to increase the accuracy of CRISPR-Cas9, since there is a possibility of removing the "healthy" gene and preserving the defective one. In addition, mutations may appear in the process of restoring crossed DNA.
The Cas9 protein crosses both strands of DNA, separating a healthy gene. Threads recover fairly quickly. The mechanism of DNA repair in a cell is based on copying a damaged gene from an identical section of an entire DNA chain. This site acts as a template that is used as a molecular blueprint, allowing the cell to more accurately reconstruct DNA.
The researchers proposed to provide cells with a donor DNA template, which will increase the accuracy in restoring the sequence of nitrogenous bases and lead to DNA repair with minimal risk of mutations. This will allow you to correct the defective gene with high accuracy.
The researchers used a modified form of the Cas9 protein (Cas9-nicase), which partially crossed only one strand of DNA. Simultaneous removal of the target gene and the gene on the donor DNA led to increased accuracy in restoring DNA integrity and achieving the desired editing result.
The researchers found that the SNGD technique they developed (single nicks in the target gene and donor plasmid, a single incision in the target gene and donor plasmid) suppresses the formation of unwanted mutations compared to the traditional CRISPR-Cas9 technique. In one of the experiments, the error rate when editing with the CRISPR-Cas9 technique was 90%, while when using the modified SNGD technique, it did not exceed 5%.
Thus, the use of donor DNA as a sample for gene repair in combination with incomplete DNA crossing significantly reduces the risk of mutations formed during genome editing. It is important to note that the increase in accuracy did not affect the performance of the SNGD method.
Article by Kazuhiro Nakajima et al. Precise and efficient nucleotide substitution near genomic nick via noncanonical homology-directed repair is published in the journal Genome Research.
Aminat Adzhieva, portal "Eternal Youth" http://vechnayamolodost.ru based on the materials of Osaka University: New genome-editing method "cuts back" on unwanted genetic mutations.