Tuesday, September 27, 2022

CRISPR-Cas9: How Bacteria Suggested New Technology to Geneticists

Время на чтение 8 мин.

In the late 1980s, Francisco Mojica was a graduate student at the Spanish University of Alicante. He studied archaea — single-celled, non-nuclear organisms — that lived in a salt marsh near the city. In the nucleus of these cells, Mojica found strange DNA sequences: unique fragments of the order of 30 nucleotides long were separated by repeating sections of the same size. At that time, no one understood why this was necessary, but later it turned out that this discovery became the first brick in the foundation of a technology that is likely to change our world.

What Doesn’t Kill a Bacterium Makes It Stronger

Later, CRISPR — Clustered Regularly Interspaced Palindromic Repeats — were found in the DNA of other archaea and bacteria. GenBank helped to understand the nature of the phenomenon. This is a database into which information about DNA and RNA sequences, as well as the proteins they encode, is loaded. It now describes almost 300 million sequences. But those who were there in 2002 were enough to find out: the CRISPR regions contain bacteriophage DNA.

Bacteriophages are viruses that infect single-celled organisms. Attaching to membranes, bacteriophages introduce particles of their DNA into the cell. They insert themselves into the DNA of the host bacterium and force it to produce the proteins needed to build new viruses. In the end, the infected organism dies, and brand-new bacteriophages go hunting for the next ones.

But, of course, there are bacteria that are stronger than the virus and emerge victorious from this deadly fight. They do not just survive, but also embed a particle of viral DNA into their own – this is CRISPR. A special Cas protein (CRISPR associated protein) is attached to it. Together, this system analyzes any DNA that is inside the bacterium. And if it turns out that it is like CRISPR, then the Cas protein cuts out this sequence. Unable to endure such an operation, the foreign DNA disintegrates, and infection does not occur.

Thus, CRISPR-Cas9 (number 9 means protein type) is responsible for the adaptive immunity of bacteria and archaea. If a single-celled organism encounters a bacteriophage but survives, it cuts the virus’s DNA into pieces and inserts it into its own. And the next time the bacterium is attacked by such a bacteriophage, it sends CRISPR-Cas9 at it.

Francisco Mojica described the purpose of CRISPR in a paper back in 2003 but could not publish it for two years – reputable scientific journals, including Nature, rejected it. Only at the beginning of 2005 did the article appear in the Journal of Molecular Evolution.

Works in Amoebas Too

The study of CRISPR-Cas9 continued, biologists clarified not only the purpose of this system, but also the mechanism of its work in wildlife. Emmanuelle Charpentier and Jennifer Downa, who in 2020 became laureates of the Nobel Prize in Chemistry, worked in this area. Once talking on the sidelines of a scientific conference in Costa Rica, scientists asked themselves the question: can CRISPR-Cas9 be adapted to work with any DNA sequence? By combining the efforts of their laboratories, the researchers found that yes. And thus became the pioneers of gene editing technology using CRISPR-Cas9.

In general, the technology is not too complicated. We take a nucleotide sequence from the target gene, connect it to the Cas protein, and launch it into the cell. The system finds this sequence and cuts the DNA – just like in the case of viral particles, but with one exception. The DNA of more complex organisms (including humans) can repair breaks. However, the gene in which this break was made can no longer function. Thus, in general, the organism continues to function, but a specific gene in it stops working. This process is called gene knockout.

Find and Replace

But what if we do not need to “turn off” the gene, but edit it? Let’s say that a mutation has accidentally occurred in it, which has led to inoperability or incorrect functioning, and we want to correct this error. This is where Magestic technology comes to the rescue — Multiplexed Accurate Genome Editing with Short, Trackable, Integrated Cellular barcodes, an article about which was published in Nature Biotechnology.

The fact is that when DNA tries to close the gap on its own, it most often happens with errors. She may lose something or vice versa, insert something superfluous. As a result, the gene will still not encode the protein as it should. To avoid this, you need to “slip” the necessary fragment on the DNA in time — then it will follow the path of least resistance and insert it. The Magestic technology just allows you to attach such a fragment to CRISPR-Cas9. The whole system works like the Find and Replace feature in Word. We set the DNA fragment to be found, set the replacement fragment, and start the process.

And this mechanism works on any organism, including mammals and humans. Of course, we don’t have to fiddle with each cell individually. The editing system can be delivered to cells using adeno-associated viruses, much like the exchange between bacteriophages and unicellular organisms occurs in nature. However, adeno-associated viruses may be associated with the risk of male infertility, so more reliable options are being developed, for example, lipid nanoparticles in which the system is enclosed in a lipid-coated capsule. After the job is done, the capsule simply dissolves.

Treatment and Punishment

The CRISPR-Cas9 technology is so simple that anyone can buy reagents for it — they are freely sold on specialized sites. With a minimally equipped laboratory, you can start your experiments.

Of course, CRISPR-Cas9 is of the greatest interest in the context of biomedicine. The technology opens the way to a simple and effective treatment for genetic diseases that are currently incurable. The CRISPR-Cas9 treatment for beta-thalassemia and sickle-cell anemia has already been successfully tested in humans.

Both diseases are caused by a mutation in the gene responsible for encoding beta-hemoglobin. With the help of CRISPR-Cas9, the scientists turned off this gene and turned on another one that codes for fetal hemoglobin (it is normally produced only in infants, and this gene does not work in adults). To perform the procedure, bone marrow cells were removed from the patients, genetic editing was carried out in them, and the altered cells were introduced back. As a result, a patient with beta thalassemia no longer required constant blood transfusions, and a patient with sickle cell anemia no longer suffered from clogged blood vessels.

If we go further, then changes in genes can be made even before birth, getting genetically modified people. Such an experiment was carried out by Chinese biologist He Jiankui in 2018. Through artificial insemination, he changed the gene of future twin girls so that they became immune to HIV. In nature, such people are found — they really have a mutation in the CCR5 gene, which makes them immune to the virus. However, the experience turned into a huge scandal. The university where Jiankui worked said it had nothing to do with the experiment. It turned out that the documents authorizing the experiment were forged, and the patients did not know that they were being implanted with genetically modified embryos. As a result, Jiankui received three years in prison and a fine of three million yuan.

Genetic editing, especially in humans, is likely to be one of the most ethically debated topics in the coming decades. This can be judged, if only because the controversy around GMO products does not subside, although they have existed for several decades and there have probably been thousands of studies proving their harmlessness.

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