Brake for CRISPR

A protein suppressing the action of the genomic editing system was tested to prevent mutations

Marina Astvatsaturyan, Echo of Moscow

Last year, two groups of scientists reported the discovery of several proteins that are able to block the activity of the CRISPR-Cas9 system. In nature, it exists in bacteria, but in recent years it has been actively used by experimenters to introduce targeted mutations.

In the July issue of the journal Science Advances, researchers from the University of California Berkeley and San Francisco (UCB and UCSF) described the use of anti-CRISPR proteins to reduce the off-target effects of genomic editing (Shin et al., Disabling Cas9 by an anti-CRISPR DNA mimic).

"CRISPR was considered a bacterial immune system, protection against viruses, and, as often happens in biology, if something is a weapon, then the target it is aimed at takes retaliatory measures," one of the co-authors of the work Jacob Corn from Berkeley explained in a comment to The Scientist. Phages – bacterial viruses – have developed anti-CRISPR proteins to fight CRISPR systems.

Studying the mechanism that works in the case of one of these proteins (it is isolated from the bacteriophage Listeria and is called AcrIIA4), the authors turned to electron microscopy and experiments in human cell culture. They managed to find out that the action of the anti-CRISPR protein is based on the connection with the Cas9 editing enzyme in a certain place, a recess in its structure, where the connection with the DNA to be cut usually occurs. The anti-protein prevents binding to DNA by filling this "pocket". 

Drawing from the UC Berkeley Anti-CRISPR proteins press release
decrease off-target side effects of CRISPR-Cas9 – VM.

The effectiveness of the anti-CRISPR system in human cell culture was tested by Korn and colleagues on the vascular endothelial growth factor A gene, which is already known for the abundance of side mutations during editing, as well as on the hemoglobin gene, which was previously used in this laboratory to correct mutations that cause sickle cell anemia.

The analysis showed that by timing the addition of the anti-protein AcrIIA4 and Cas9 itself, it is possible to control the blocking of non-targeted effects. "We found that if you add an anti-protein first, and immediately after it Cas9, which cuts DNA, it will cause complete prevention of editing, but if you delay adding Cas9 for about six hours, then the targeted editing will not suffer much, and the side effect will be much less," says Korn.

Let me remind you that in early June, a report about a study was widely distributed, in which it was shown that the use of the CRISPR genomic editing technique is associated with a larger number of unforeseen mutations than expected, and this caused panic among investors, as well as criticism of the study for the lack of correct control samples.

Portal "Eternal youth" http://vechnayamolodost.ru  20.07.2017