Genetic engineering opens up opportunities for humanity to create previously non-existent organisms and to destroy genetic diseases. However, things are not so rosy, since even the breakthrough CRISPR / Cas9 technology is far from perfect. The mistakes she makes may be rare, but one is enough to become fatal to a person. Lenta.ru talks about what is wrong with CRISPR and how scientists are trying to fix the situation.
The CRISPR / Cas9 system - a kind of DNA scissors - is rightfully considered a revolution in the field of genetic engineering. With its help, scientists can edit the human genome, removing harmful mutations from it, and thus treat unpleasant and deadly hereditary diseases. One should not think, however, that there were no such methods before. In the arsenal of geneticists were, for example, nucleases containing zinc "fingers", and endonucleases - enzymes that break DNA molecules in specific places. In terms of accuracy, versatility and cost, they are noticeably inferior to CRISPR / Cas9, although the latter is far from perfect.
CRISPR / Cas9 was originally created not by scientists, but by nature. It is a molecular mechanism that exists inside bacteria and allows them to fight bacteriophages and other parasites. In fact, it works as an immunity against infection. CRISPR (stands for "short palindromic repeats, regularly spaced in groups") are special regions (loci) of DNA. They contain short fragments of DNA viruses that once infected the ancestors of today's bacteria, but were defeated by their internal defenses. These pieces are called spacers and are separated from each other by repeating sequences.
When a bacteriophage invades a bacterium, each repeating sequence and adjacent spacer are used as a template for the synthesis of molecules called crRNAs. Many different RNA chains are formed, they bind to the Cas9 protein, whose task is extremely simple: to cut the DNA of the virus. However, he will be able to do this only after crRNA finds a complementary fragment of viral DNA. After Cas9 breaks apart the foreign nucleic acid, the latter is completely destroyed by other nucleases.
CRISPR / Cas9 is good precisely for its accuracy, because for bacteria the correct functioning of the immune system is a matter of life and death. The "anti-virus" system needs to find a section of viral DNA among a million others and, most importantly, not to confuse it with its own genome. Over millions of years of evolution, bacteria have perfected this mechanism. So right after they figured out why a CRISPR system was needed, they realized that it could be tamed as an unprecedentedly accurate gene editing tool.
To replace one specific region in the genome with another, it is necessary to synthesize guide RNA, which is similar in principle to crRNA. She tells Cas9 where it is necessary to make a double-strand break in the DNA of the modified organism. However, we do not need to spoil the gene, but modify it - for example, replace one or more nucleotides and remove the harmful mutation. Here nature comes to the rescue again. Natural repair mechanisms immediately begin to restore the cut chain. The trick is that for this, some RNA fragments are removed near the break, after which similar sequences are inserted there. Scientists can replace them with their own DNA sequences and thus modify the genome.
Schematic representation of CRISPR
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Image: Kaidor / Wikipedia
However, nothing is perfect. Despite the relative accuracy, CRISPR systems sometimes make mistakes. One of the reasons lies in the very nature of the system. It is disadvantageous for bacteria for crRNA to coincide by 100 percent with a fragment of viral DNA, which may differ by one or two nucleotides. It is better for her that some nucleotides could be different, which gives the microorganism a better chance of fighting the infection. At the same time, in genetic engineering, low specificity threatens with errors: changes can be made in the wrong place. If this happens in the course of experiments on mice, then there will be no special tragedy, but editing the human genome could turn into a disaster.
This explains the concern of Western scientists about the experiments that are being carried out in China. Asian researchers have used CRISPR technology to genetically modify human embryos. Such experiments have been banned in Europe and the United States, but recently the UK has allowed them - solely for research purposes. Such embryos will have to be destroyed in a couple of weeks after receiving, which excludes the "breeding" of GM people.
However, CRISPR / Cas9 wouldn't be so great if it couldn't be improved. So, scientists taught Cas9 to cut not two chains at once, but only one. The cut is made at two different places in the DNA sequence on different strands, so the system must be able to recognize twice as many nucleotides as normal, making it more accurate.
Protein Cas and crRNA
Photo: Thomas Splettstoesser / Wikipedia
Scientists at the University of Western Ontario have found another way to improve this technology. They were trying to solve the problem of repairing the cut DNA. The rapid restoration of the nucleic acid chain leads to the fact that scientists do not have time to make their own corrections to the genome. Thus, a vicious circle is created: the chain, repaired in an undesirable way, has to be cut again with the Cas9 protein.
To prevent this from happening, the researchers modified the protein scissors to create the TevCas9 protein. It cuts the DNA strand in two places, making it difficult to repair the site. To synthesize the new enzyme, the enzyme I-Tevl was added to Cas 9, which is also an endonuclease, that is, a protein that cleaves a DNA molecule in the middle, rather than cleaving off the ends of the sequence, as exonucleases do. The resulting fusion protein turned out to be more accurate in binding to specific sites and less likely to make a mistake and cut the wrong site.
Crystal structure of Cas9 bound to DNA
Photo: Cas9 wiki project / Wikipedia
There is another way to improve the accuracy of CRISPR systems. The "arms race" between bacteria and viruses has led not only to the development of defense systems in microorganisms, but also to ways of neutralizing them. Thus, bacteriophages mutate rapidly, losing the areas by which bacterial immunity recognizes them. However, some code for anti-CRISPR proteins, interfering with the work of the crRNA-Cas9 complex.
On December 8, the journal Cell published an article by scientists from the University of Toronto who created "anti-CRISPR" - a system that allows you to turn off the mechanism under certain conditions. It prevents unwanted errors by suppressing Cas9 activity in the event that the guide RNA binds to the wrong fragment. Anti-CRISPR consists of three proteins that inhibit nuclease and are encoded by the genes of one of the bacterial viruses.
Already, CRISPR technology is being used to treat serious diseases such as leukemia and lung cancer, and is also being tested to cleanse immune cells of HIV. As scientists find new ways to improve this method, more and more opportunities for its application will open up.
Alexander Enikeev