How to fix a person
CRISPR/Cas9: The latest gene modification system that promises to change our lives
Sergey Vasiliev, Naked Science
Fantastic, frightening stories about interference in the human genome have remained fantastic for many years. But now there is such a method: the CRISPR/Cas9 system borrowed from bacteria allows genetic modification of any organisms with very high accuracy.
Immunity
– the natural "immunity" of bacteria, a biochemical system of protection against viruses, which is required by single-celled organisms that are unable to maintain such a complex immune system as ours. The first hints of its existence were found back in the late 1980s, when Yoshizumi Iishino and his colleagues studied the common E. coli, more precisely, one of its unremarkable gene (iap).
Just in case, the Japanese sequenced its sequence along with the sections on the sides of it: maybe there will be some fragments involved in the regulation of iap activity?.. Instead, biologists found in DNA long sequences of repetitive, completely identical repeats with a length of exactly 29 nucleotides. Between them – like dry plants between sheets of paper in a herbarium – there were "laid" short fragments with a length of 32 nucleotides, which were not repeated in any way.
The structure of nucleotides / ©wikipedia
Later, this strange part of DNA was called "regularly grouped, separated short palindromic repeats" – Clustered Regularly Interspaced Short Palindromic Repeats. Otherwise, work on them stopped for a long time, although many scientists became interested in the mysterious sections of the chromosome, and some even speculated 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," Iishino and co–authors wrote at the time.
However, in the second half of the 1990s, a real sequencing boom began. It became easier and easier to establish the DNA sequence, and the genomes of more and more new organisms began to replenish computer databases and be analyzed from all sides. The mysterious – and seemingly meaningless, completely unlike any gene – CRISPR sequence was found in bacteria everywhere. Dutch biologist Ruud Jansen noticed that they are always adjacent to the genes of the same proteins. Their functions were also unknown at that time, and they were simply called "CRISPR-Associated Proteins" (Cas).
Simplified diagram of the structure of CRISPR / ©wikipedia
And only in 2005, three groups of researchers reported at once that the unique CRISPR sites are fragments of viral genomes. "Something clicked here," the world–famous bioinformatician and evolutionist Evgeny Kunin later recalled. By that time, he had been struggling with the riddle of CRISPR for several years – and finally it dawned on him: this DNA may be part of the antiviral defense of a bacterial cell.
This idea appealed to microbiologist Rodolphe Barrang, who at that time worked at 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 Streptococcus thermophilus with two strains of bacteriophages. Most of the bacteria died, but the survivors turned out to be quite resistant to these viruses. Sequencing their DNA, the scientists confirmed that traces of the meeting appeared in it.
The tool
Jennifer Dudna and Blake Wiedenheft took up the study of the structure of Cas proteins: by this time it turned out that they perform the role of nucleases, that is, they cut DNA. Despite all the findings, the significance of the discovery was still unclear: "You don't have any specific practical purpose," Dudna explained to Wiedenheft, who worked in her laboratory. "It's just important to understand how it works." But as we worked, 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 it or its ancestors had to deal with. Special proteins associated with CRISPR (CRISPR-Associated Proteins, Cas) can use this catalog. Focusing on these samples, they quickly recognize new viral genes and cut them, disabling them.
Biologist Karl Zimmer explains the work of the CRISPR/Cas system as follows: "As the CRISPR region is filled with viral DNA, it becomes a key "gallery" in the cell, where "portraits" of microbes that bacteria have encountered are presented. Subsequently, this viral DNA can be used to "guide" the precise weapon of Cas proteins."
To do this, the bacterial cell synthesizes short samples, RNA molecules, on the preserved DNA fragments. Each of these RNA "guides" (gRNA) binds to a Cas protein capable of cutting DNA suitable for this sample. These complexes constantly patrol the cell, tracking the appearance of any DNA and matching 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, an interesting moment occurred," Jennifer Dudna later recalled. "We exclaimed, 'God, this could be a tool!'"
Today, a whole family of Cas proteins has been identified, but the most studied and mastered protein was Cas9, isolated from Streptococcus pyogenes bacteria – pathogens of scarlet fever. It was he who formed the basis of the latest technique of genetic modification of living organisms CRISPR/Cas9, a technique that promises an unprecedented breakthrough in biotechnology, agriculture and medicine.
Palindromes in DNA: A. Palindrome, B. Ring, C. Stem / ©wikipedia
Modification
In fact, the Cas9 protein is a nuclease, that is, an enzyme that cuts 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.
Recall 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 about centimeters. This polymer does not differ in diversity, consisting of only four different units: adenine (A), guanine (G), thymine (T) and cytosine (C), which are repeated millions and millions of times. It is incredibly difficult to find exactly the right area in this monotony.
Crystal structure of S. aureus Cas9 in complex with SRNA and its target DNA / ©wikipedia
For a long time, geneticists had at their disposal only systems with nucleases that recognized short sections – for example, four nucleotides of ATCC or THCA – of which dozens and hundreds can occur throughout the chain. As a result, the incisions were made in a random one of these places, and only painstaking work allowed us to select the cells in which this process took place in the right part of the genome. In contrast, the Cas9 protein armed with gRNA recognizes a fragment the length of this RNA – about 20 nucleotides. Such sites are already, as a rule, 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 desired organism in a computer, find a fragment that should be cut, and synthesize gRNA molecules with the same sequence of bases (and thymine replacement, whose role in RNA is played by uracil, Y). Cas9 nucleases are illegible and will cut DNA anywhere, as long as the gRNA matches.
Crystal structure of Cas9 bound to DNA / ©Nature
In contrast, the systems of genetic modification of previous generations required a long work on the design and synthesis of enzymes-nucleases capable of recognizing certain parts of DNA. For example, methods using DNA-binding "zinc fingers" ZFN (Zinc Finger Nuclease) or TALEN (Transcription Activator-Like Effector Nucleases) proteins theoretically allow you to work with even longer DNA fragments. However, they have to be designed separately for each specific task.
Finally, CRISPR/Cas9 is universal in relation to different types of modified organisms. The method is simple and effective and, at least theoretically, 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 we move on to people, let's "practice on cats." And better – on mice.
Mice, people and everything-everything-everything
The example with the "albinism gene" is extremely unfortunate: there is no such gene – there is (as it is written in the next paragraph) the absence or blockade of the tyrosinase enzyme, and albino mice have long been bred by conventional breeding, and how albinism affects human health has long been known. But make claims to the author of the article reprinted here – VM.
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 melanin pigment. 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.
To begin with, we should 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 – the exchange of homologous sections of chromosomes – so, after spitting three times, let's hope that it will capture the fragment we need. By trial and error, endless repetitions and rejection, we can get mice that have 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's better to switch to CRISPR/Cas9 right away.
Indeed, in order to get the same albino mice, it is enough to find the border areas of our target gene and synthesize gRNA for them, after which it is introduced into the embryo along with Cas9 proteins and DNA of the new gene. Picking up the gRNA, Cas9 nucleases will cut both copies of the gene at the edges, after which cellular repair systems responsible for maintaining the integrity of the genome will be involved.
This is an extremely responsible task, so the repair proteins act quickly and even roughly. Having discovered DNA damage – especially such a dangerous one as a double–stranded incision - they are ready to pick up the first piece of DNA that comes along, literally "plugging" the gap that has formed. So if there are enough fragments we need in the cell, they will be embedded in the place cut by Cas9 proteins.
It is not for nothing that, since the discovery of CRISPR/Cas9, genetic modification has been making breakthrough after breakthrough. The loud statement of Chinese biologists is just one example. China 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 at the age of no older than 14 days. In China, much more is allowed.
Such work is incredibly promising: literally in recent years, it has been shown that CRISPR/Cas9 allows you to edit genes even in an adult body, purifying the DNA of T-lymphocytes from the HIV that infected them. And in the same China, scientists (not too successfully) tried to get embryos resistant to this infection. Now we are talking about the fight against cancer. To do this, doctors plan to edit the DNA of the same T-lymphocytes – more precisely, the gene of the PD-1 protein, which normally keeps them under control.
The structure of the human immunodeficiency virus / ©wikipedia
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, such an ability would be very appropriate, and scientists are going to take cells from real cancer patients and change the PD-1 gene using CRISPR/Cas9 (now we understand in general terms how this can be done). Having returned these lymphocytes to the body, the authors expect that they will begin 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 to get rid of many other diseases. Moreover, many of the most serious conditions are associated with a malfunction of just one gene: it will probably be much easier to fix them than to cure the same cancer. In contrast, kindness and intelligence, beauty or athletic abilities are the product of the work of a lot of different genes, upbringing and other environmental factors. So CRISPR/Cas9 will only benefit, and it is unlikely to be used to harm. Unless it's just to scare.
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