How to fix a person. CRISPR / Cas9: the latest gene modification system that promises to change our lives

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How to fix a person. CRISPR / Cas9: the latest gene modification system that promises to change our lives
How to fix a person. CRISPR / Cas9: the latest gene modification system that promises to change our lives

Fantastic, frightening to many stories about interference with the human genome for many years remained just fantastic. There were no practical methods to change DNA, with good intentions or otherwise. But now there is such a method: the CRISPR / Cas9 system borrowed from bacteria makes it possible to carry out genetic modification of any organisms with such precision and efficiency that one could only dream of before. And now, Chinese doctors have announced plans for the first targeted editing of human DNA. Intervention in our genome is no longer a fantasy. But is it really scary?



- the natural "immunity" of bacteria, a biochemical system of defense against viruses, which is required by unicellular organisms that are unable to support such a complex immune system as ours. The first hints of its existence were found back in the late 1980s, when Yoshizumi Ishino and his colleagues studied the common E. coli, more precisely, one of its unimportant gene (iap).

Just in case, the Japanese sequenced its sequence along with the regions on its sides: maybe there will be some fragments involved in the regulation of iap activity?.. Instead, biologists found in DNA long sequences of repeating, completely identical repeats exactly 29 nucleotides in length. Between them - like dry plants between sheets of paper in a herbarium - short fragments of 32 nucleotides in length were "laid", which did not repeat in any way.


This strange piece of DNA was later called Clustered Regularly Interspaced Short Palindromic Repeats. Otherwise, work on them stopped for a long time, although many scientists became interested in the mysterious regions of the chromosome, and some even talked about their role. The functional significance of CRISPR remained a mystery, and no one expected any special breakthroughs from them: "The biological significance of these sequences is unclear," Isino et al. Wrote at the time.

However, in the second half of the 1990s, a real sequencing boom began. It became easier to establish the DNA sequence, and the genomes of more and more new organisms began to replenish computer databases and were analyzed from all sides. Mysterious - and seemingly meaningless, completely unlike any gene - the CRISPR sequence has been found everywhere in bacteria. The Dutch biologist Ruud Jansen noticed that they always coexist with the genes of the same proteins. Their functions were then also unknown, and they were simply called "proteins associated with CRISPR" (CRISPR-Associated Proteins, Cas).


And only in 2005, three groups of researchers at once reported that the unique regions of CRISPR are fragments of viral genomes. “Then something clicked for me,” the world famous bioinformatist and evolutionist Evgeny Kunin later recalled. By that time, he had been struggling for several years over the riddle of CRISPR - and, finally, it dawned on him: this DNA could be part of the antiviral defense of a bacterial cell.

The idea appealed to microbiologist Rodolphe Barrang, who at the time worked for Danisco, a yogurt company.In this business, a viral epidemic among lactic acid bacteria can cause serious losses, and the researcher was looking for methods to protect against it. To test Kunin's hypothesis, he infected Streptococcus thermophilus with two strains of bacteriophages. Most of the bacteria died, but the survivors were quite resistant to these viruses. By sequencing their DNA, the scientists confirmed that traces of the meeting appeared in it.


Jennifer Doudna and Blake Wiedenheft undertook to study the structure of Cas proteins: by this time it turned out that they act as nucleases, that is, they cut DNA. Despite all the findings, the significance of the discovery was still unclear: “You have no definite practical purpose,” Dudna explained to Wiedenheft, who worked in her laboratory. “It’s just important to understand how it works.” But as the work progressed, many amazing details came to light.

CRISPR is, indeed, something like a herbarium, a catalog in which a bacterial cell stores samples, fragments of the genomes of viruses that she or her ancestors had to encounter. Special proteins associated with CRISPR (CRISPR-Associated Proteins, Cas) can use this catalog. Based on these patterns, they quickly recognize new viral genes and cut them, incapacitating them.


Biologist Karl Zimmer explains the CRISPR / Cas system as follows: “As the CRISPR region fills with viral DNA, it becomes a key gallery in the cell, displaying portraits of microbes that bacteria have encountered. Subsequently, this viral DNA can be used to "target" the precise weapon of Cas proteins."

For this, the bacterial cell synthesizes short samples, RNA molecules, on the stored DNA fragments. Each of these RNA guides (gRNAs) binds to the Cas protein, which can cut DNA matching the sample. These complexes constantly patrol the cell, tracking the appearance of any DNA and comparing it with gRNA. If there is a match, the DNA double helix is ​​immediately cut into pieces and inactivated. “As soon as we realized Cas as programmable, DNA-cutting enzymes, it was an interesting moment,” Jennifer Dudna later recalled. - We exclaimed: "God, this can be an instrument!"

Today, a whole family of Cas proteins has been identified, but the Cas9 protein, isolated from the bacteria Streptococcus pyogenes, the causative agents of scarlet fever, turned out to be the most studied and mastered. It was he who formed the basis of the newest method of genetic modification of living organisms CRISPR / Cas9, a method that promises an unprecedented breakthrough in biotechnology, agriculture and medicine.



Indeed, the Cas9 protein is a nuclease, that is, an enzyme that cleaves DNA. For any method of genetic modification - removing or adding targeted active genes to the body - this ability plays a key role. To copy and paste, you need to cut, and do it in a strictly defined place. Until now, geneticists have had problems with accuracy.

Let's remember that a DNA molecule is, by molecular standards, an incredibly long chain, the total length of which in each chromosome of each of our cells reaches the order of centimeters. This polymer does not differ in variety, consisting of only four different units: adenine (A), guanine (G), thymine (T) and cytosine (C), which are repeated millions and millions of times. Finding the right site in this monotony is incredibly difficult.


For a long time, geneticists had only systems with nucleases that recognized short regions - for example, four nucleotides ATTSC or THCA - of which tens and hundreds can occur along the chain. As a result, the cuts were made at random from these places, and only painstaking work allowed us to select the cells in which this process took place in the desired region of the genome. In contrast, the Cas9 gRNA-armed protein recognizes a fragment with a length of this RNA - about 20 nucleotides. Such regions, as a rule, are not repeated at all in the DNA of even higher organisms.

Moreover, the very structure of the Cas9 complex with gRNA determines the ease of working with it. It is enough to open a database with the DNA of the organism in question in a computer, find a fragment that must be cut, and synthesize gRNA molecules with the same base sequence (and replacement of thymine, the role of which in RNA is played by uracil, U). Cas9 - nucleases are illegible and will cut DNA anywhere as long as the gRNA matches.


In contrast, the systems of genetic modification of previous generations required lengthy work to design and synthesize nuclease enzymes capable of recognizing specific regions of DNA. For example, methods using DNA-binding zinc fingers ZFN (Zinc Finger Nuclease) or TALEN proteins (Transcription Activator-Like Effector Nucleases) theoretically allow working with even longer DNA fragments. However, for each specific task, they have to be designed separately.

Finally, CRISPR / Cas9 is universal in relation to different types of modified organisms. The method is simple and effective and, at least in theory, is equally suitable for obtaining rice with a high content of vitamin A or salmon gaining weight twice as fast as usual, for introducing new genes or replacing defective ones in breeding horses and humans … But before moving on to people, let's "practice on cats." And better - on mice.

Mice, people and everything-everything-everything

Let's imagine that we need to get albino mice in order to study how this condition affects the health of different body systems in humans. To do this, you should "turn off" both copies of the gene associated with the synthesis of the pigment melanin. If we are committed to traditional approaches to genetic modification (by the way, for the most part, also borrowed from bacteria), then we should be patient.

First, we need to synthesize the "albino gene" and get mouse embryos at the very first stages of development. Then, new DNA is introduced into their nuclei through the thinnest hollow glass needle. In dividing cells, recombination occurs - an exchange of homologous regions of chromosomes - so, after spitting three times, let's hope that it will capture the fragment we need. Through trial and error, endless repetition and culling, we can get mice that received one copy of the "albino gene" and were able to pass it on to their offspring. Then, by crossing such animals, sooner or later we will achieve the birth of individuals with the replacement of both copies. You can wait, but it is better to immediately switch to CRISPR / Cas9.


Indeed, to get the same albino mice, it is enough to find the border regions of our target gene and synthesize gRNA for them, and then introduce a new gene together with Cas9 proteins and DNA into the embryo. Having picked up the gRNA, Cas9 nucleases will cut both copies of the gene along the edges, after which the cellular repair systems responsible for maintaining the integrity of the genome are involved.

This is an extremely demanding task, therefore, repair proteins act quickly and even rudely. Having discovered DNA damage - all the more so dangerous as a double-stranded cut - they are ready to pick up the first piece of DNA they find, literally "plugging" the gap. So if there are enough fragments in the cell, they will be inserted into the place cut by Cas9 proteins.

It is not without reason that genetic modification has made breakthrough after breakthrough since the discovery of CRISPR / Cas9. The loud statement of Chinese biologists is just one example. The PRC remains a country with one of the softest laws in the field of genetic engineering. Even in the UK, where experiments on the use of CRISPR / Cas9 on human embryos are allowed, the resulting chimeras must be destroyed before the age of 14 days. Much more is allowed in China.

Such works are incredibly promising: literally in recent years, it has been shown that CRISPR / Cas9 makes it possible to edit genes even in an adult organism, clearing the DNA of T-lymphocytes from the HIV infecting them. And in the same China, scientists (not very successfully) tried to get embryos resistant to this infection.Now it's about fighting cancer. To do this, doctors plan to edit the DNA of the same T-lymphocytes - more precisely, the gene for the PD-1 protein, which normally keeps them under control.

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The active PD-1 gene blocks the ability of T lymphocytes to attack the body's own cells and prevents the development of autoimmune diseases. However, in the case of cancer, this ability would be very appropriate, and scientists are going, taking cells from real cancer patients, to change the PD-1 gene using CRISPR / Cas9 (now we understand in general terms how this can be done). By returning these lymphocytes to the body, the authors expect them to multiply and attack the tumor.

Cancer and HIV are just a couple of high-profile examples. However, in the future, CRISPR / Cas9 and genetic modification will help get rid of many other diseases. Moreover, many of the most serious conditions are associated with a disruption in the work of just one gene: they, apparently, will be much easier to fix than to cure the same cancer. In contrast, kindness and intelligence, beauty or athletic ability are the product of the work of a mass of different genes, upbringing and other environmental factors. So CRISPR / Cas9 will only be beneficial, and it is unlikely that it will be possible to use it to harm. Is that just to scare.

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