Gen, what's wrong with you?

CRISPR-Cas9 technology attracts a lot of attention from both scientists and all those interested in biotechnology. Many believe that a new method of precise gene editing will create the perfect human in the future.

In early February 2016, it became known that the UK government had allowed scientists to alter the DNA of human embryos for research purposes using the CRISPR system. We are not talking about the creation of GMO-people, since all modified embryos obtained through in vitro fertilization will be destroyed after 14 days.

However, the public was greatly concerned. For example, the director of US national intelligence, James Clapper, said that potentially genome editing technologies are weapons of mass destruction. His pessimistic forecast was embodied in the new season of the series "The X-Files", where the CRISPR system was used for global genocide. What is CRISPR technology, why does it cause so much excitement among scientists, fears among the public, and what can it really give to humanity?

Antivirus protection

CRISPR is the immune system of bacteria and archaea that rescues microorganisms from viruses. It was first discovered by Japanese scientists in the late 1980s in the bacterium Escherichia coli (Escherichia coli). They noticed that the bacterial genome contains repetitive sequences separated by spacers - unique regions. However, what role all this performs, then they could not figure out. A similar genetic cassette structure was found later in another microorganism - the archaea Haloferax mediterranei, and then in many other prokaryotes. Such areas became known as the acronym CRISPR, that is, Clustered Regularly Interspaced Short Palindromic Repeats. In Russian - "short palindromic repetitions, regularly arranged in groups."

More than ten years later, geneticists have established that next to CRISPR cassettes are located genes that encode proteins called Cas. Known spacers have been compared to DNA sequences from extensive genomic databases. It turned out that the spacers are very similar to the regions of the genomes of bacteriophage viruses, as well as plasmids - circular DNA molecules usually found in bacteria.

A group of bioinformatics led by Evgeny Kunin from the National Center for Biotechnological Information proposed the mechanism of operation of CRISPR cassettes and associated Cas proteins. A virus that has entered a bacterial cell is detected by a complex of Cas proteins carrying a spacer sequence. If the latter coincides with a section of the virus' DNA (protospacer), then Cas proteins cut the foreign DNA, preventing infection. Later, scientists were able to insert a spacer with a fragment of the bacteriophage genome into the bacteria's CRISPR cassette and observed how the microorganism successfully coped with the virus. This served as one of the proofs of the proposed hypothesis.

Spacers in CRISPR cassettes are a template for the production of crRNA, which is sent along with Cas proteins to attack the virus. Where do spacers come from? When a bacterium encounters an unknown virus, it begins to cut various pieces of DNA from its own and foreign genomes and insert them into the cassette. Of course, most of these pieces are useless and even harmful, but the one that helps the body fight the infection remains in CRISPR and is passed on to the descendants of the bacteria.

Penetrating the holy of holies

It turned out that there are several varieties of the CRISPR-Cas system. One of them encodes not the Cas protein complex, but only one - Cas9. This is a universal molecule that performs several functions at once: it binds foreign DNA and cuts it. It was in the system with the Cas9 protein that scientists saw an accurate genome editing tool. In an article published in the journal Science in 2012, Emmanuelle Charpentier and Jennifer Doudna proposed artificial sequences as crRNAs that would recognize certain sections of DNA. Then Cas9 would make cuts where scientists need it. Another research group around the same time showed that the CRISPR-Cas9 system can work with genomes not only in bacteria, but also in cells of other organisms, including humans.

And before the CRISPR system, genome editing techniques were known. For example, with the help of nucleases containing zinc fingers. These are artificial enzymes that do not exist in nature and are capable of cleaving the DNA chain. Zinc finger is a special protein module that includes one or more zinc ions. It is with the help of such structures that enzymes interact with DNA, RNA and other molecules. Scientists have connected a zinc finger to another module that cuts the DNA strand. Such nucleases can target specific regions of the genome, where cuts are made. The problem is that for each site where a break needs to be made, a specific protein must be synthesized, isolated and tested. In addition, the use of nucleases is associated with a high probability of errors: often breaks occurred in the wrong places.

The CRISPR-Cas system is much more convenient. The function of the cut is taken over by the Cas9 protein, which is the same for any target loci. All that needs to be done is to synthesize a crRNA that will tell the protein exactly where to make a double-strand break. Once the break is made, DNA repair systems are activated. First, it is the mechanism of non-homologous end joining (NHEJ), which results in various mutations that disrupt gene functions. If you make many of these breaks, you can achieve the rearrangement of a large section of DNA.

Second - homologous recombination (HR), when similar or identical DNA regions exchange nucleotide sequences with each other. This mechanism is used to repair damage to a double strand called double strand breaks.

When it comes to guided DNA editing, homologous recombination is more suitable for scientists. With the CRISPR-Cas system, breaks can be made to remove an entire region from the DNA. At the same time, geneticists slip the sequence they have created, which is embedded in the place of the deleted one. Thus, it is possible to "repair" mutations that cause serious diseases. Scientists remove the defective section of the gene and replace it with a normal one. Moreover, it is possible to introduce new mutations, create different variants of the same gene, add specific sequences to it, which affects the functions of the protein encoded by it.

Many defective genes can be corrected at once. To do this, you only need to synthesize the corresponding crRNA, whose sequences coincide with the required DNA regions. Cas9 proteins bind to crRNA and strive to “repair” genes. It should be clarified that when we talk about a match, we mean a complementary match. The principle of complementarity shows in which case bonds will form between different strands of DNA or RNA. Nucleotide A binds to nucleotide T, and nucleotide C to G. Therefore, for example, the ACTG fragment matches TGAC.

Weapons Against Disease

When it became clear that the CRISPR system could be used to edit the human genome, many laboratories around the world began active research. For example, they use technology to create genetically modified organisms. One of the directions is the creation of lactic acid bacteria that could resist the attack of bacteriophages that destroy the cultures of beneficial microorganisms. But perhaps one of the most interesting uses of CRISPR is in the fight against retroviral infections.

Retroviruses - such as HIV - insert their genome directly into the DNA of an infected cell. The journal Scientific Reports has published a paper demonstrating how the use of CRISPR-Cas9 can purify HIV-infected T-lymphocytes and even prevent the re-insertion of the virus. Geneticists simply introduced genes encoding crRNA and Cas9 into the culture of T cells, which, in turn, successfully excised the virus DNA from the lymphocyte genome.

Chinese scientists conducted experiments on human embryos even before such studies were allowed in the UK. In April 2016, geneticists reported that they had altered the genes of the embryos to make them immune to HIV. Using CRISPR, they introduced a gene that is found in people who are immune to infection.

The CRISPR system has also come in handy in the fight against cancer. For example, in a work published in Nature Biotechnology, it was shown that using a modified Cas9 protein, you can turn off certain genes and thereby determine their role in the transformation of normal cells into malignant ones. If it turns out that a mutation in a particular gene contributes to the development of cancer, then the next step is to correct the defect using genetic manipulation.

CRISPR can help treat blood cancer - leukemia. Instead of looking for a bone marrow donor, you can take tissue samples from the patient's hematopoietic organ, correct defective stem cells, rid them of the fatal mutation, and then transplant them back. If the malignant cells remaining in the diseased body are destroyed by radiation, the repaired cells will be able to multiply and produce healthy blood cells.

Pandora's Box

Is CRISPR Dangerous? At the current level of development, no. Fears are largely related to the fact that it is too early to edit the human genome in order to treat hereditary diseases. The technology is still crude. Thus, the work of Chinese scientists has been criticized for the large number of DNA breaks that have arisen in the wrong place. In addition, only a few of the fifty embryos had a correct replacement of the gene site.

If genome editing technology will save humanity from hereditary diseases, cancer, viruses, then this is a matter for the future, which, perhaps, is much further than optimists think. When it comes to creating better people and the ethical issues that come with it, this is generally beyond what CRISPR can do.