Gene editing technology using the CRISPR-Cas9 system can lead to many unwanted mutations. This conclusion was reached by scientists from the Columbia University Medical Center. "Lenta.ru" talks about a new study, the results of which were published in the journal Nature Methods.
CRISPR / Cas9 is a molecular mechanism that exists inside bacteria and allows them to fight viruses with bacteriophages. CRISPRs are "cassettes" within the DNA of a microorganism, which are made up of repeating sections and unique sequences called spacers. The latter are fragments of viral DNA. In essence, CRISPR can be thought of as a databank containing information about the genes of pathogenic agents. The Cas9 protein uses this information to correctly identify foreign DNA and render it harmless by cutting at a specific location.
The accuracy of CRISPR / Cas9 allows it to be used as a gene editing tool that is much more effective than previously existing methods. The researchers insert a DNA cassette containing the Cas9 protein gene and a region coding for a special targeting RNA molecule (sgRNA) into the cell, in whose genome changes are to be made. The latter can attach to a specific location in the genome, indicating to Cas9 where to cut. You can then insert the desired gene or group of genes into the gap, creating a modified cell.
The advantage of CRISPR / Cas9 is that it can make precise changes, reducing the number of unwanted cuts (off-target effects) in other parts of the genome. In addition, it saves time and money - unlike other widely used methods that use nuclease enzymes. The CRISPR system is more suitable for situations where several defective genes need to be corrected at once, since it only requires synthesizing the corresponding sgRNA. In the case of nucleases, for each specific DNA site, it is necessary to assemble its own specific enzyme, which is longer and more expensive.
CRISPR / Cas9 Artistic Image Credit: Steve Dixon / Feng Zhang / MIT
The technology quickly became popular, not only among scientists, but also in popular culture. For example, it appears in the plot of some episodes of the new season of The X-Files as a weapon of mass destruction. The director of US national intelligence, James Clapper, also expressed his concern, who believes that genetic engineering is a threat. However, any technology is dangerous if used incorrectly. For example, the inept and frequent use of antibiotics, which have saved the lives of hundreds of thousands of people, is fueling the emergence of antimicrobial-resistant superbugs.
Despite the fiction, CRISPR / Cas9 by itself, like antibiotics, cannot be used as a weapon of destruction of people. But you can create modified organisms, such as mosquitoes, that will spread the defective gene in their population, which can lead to a strong decrease in the number of blood-sucking insects. Biologists are urging to be extremely careful about such applications.
Immune cells attack cancerous growth Photo: Steve Gschmeissner / Science Photo Library
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At the same time, many biotechnologies believe that the technology will make it possible to successfully treat dangerous diseases, including those caused by genetic causes. For example, at the end of 2016, China had already experimented with treating aggressive lung cancer. The patient's immune cells were removed and the gene that suppresses the fight against malignant tumors was turned off. The genome modification procedure was successful, and the cells were reintroduced into the patient's body.
CRISPR / Cas9 has its drawbacks. Thus, DNA broken by molecular "scissors" loses part of the sequence, which prevents "point" editing, that is, the replacement of one single nucleotide. Biotechnologists are creating improved versions of the system by overcoming these limitations. For example, South Korean researchers have combined the Cas9 enzyme with another protein, cytidine deaminase. This taught CRISPR / Cas9 to make single nucleotide substitutions.
However, there is another problem. The fact is that this technology can also introduce unwanted mutations, and scientists do not yet fully understand how this can be eliminated. When it comes to treating people, the risk is still unreasonably high: side changes in the genome can greatly harm health.
Certain algorithms can predict where in the genome unnecessary replacements can be made. They work very well when CRISPR modifies individual cells or tissues, but are not used when it comes to living organisms.
CRISPR / Cas9 structure Photo: Ian Slaymaker / Broad Institute
For example, biotechnologists at Columbia University have used technology to remove mutations that trigger retinitis pigmentosa. For this, skin cells were taken from the patient and reprogrammed into stem cells. The defect was then corrected using a CRISPR cassette. Such modified cells can be converted into retinal cells and transplanted into a patient to restore vision.
In addition, the same scientists created genetically modified mice, which were injected with CRISPR at the zygote stage. The researchers then sequenced (determined the nucleotide sequence) the entire genome of the mice. Although CRISPR successfully corrected a gene in which the defect caused blindness, it also introduced over 1,500 single nucleotide mutations and over 100 deletions (deletions of short sections) and insertions. None of these unwanted changes were predicted by computer algorithms.
Genome modification is a good way to treat hereditary diseases that are currently incurable. According to Stephen Tsang, he and his co-authors believe it is important for genetic engineering scientists to be aware of the potential dangers of unwanted mutations that arise when editing DNA. These mutations include both nucleotide substitutions in the coding regions of the genome, and in non-coding regions, where regulatory elements are located (they control the activity of genes).
Despite the mutations, the mice did well. Biotechnologists remain optimistic about gene therapy since any medical intervention has side effects. You just need to know what they are. In the future, this will improve the technology to minimize off-target effects.
Alexander Enikeev
