23 December 2016

CRISPR/Cas9: results of the year

Woke up the devil


Scientists have high hopes for CRISPR/Cas9 technology, which makes it possible to make changes to the genomes of living organisms, including humans, with high accuracy. New scientific articles describing various types of CRISPR systems, as well as their modifications, are being published. "The tape.<url>" tells about the discoveries in this area made in 2016.

CRISPR/Cas9 is a bacterial adaptive immunity system that allows microorganisms to fight viruses. It consists of spacers – sections of DNA corresponding to certain fragments (protospacers) The DNA of the infectious agent. Spacers encode specific sgRNA molecules that bind to the Cas9 enzyme. The resulting complex is attached to the DNA chain of the virus, and Cas9 works like scissors, cutting it.

In fact, CRISPR/Cas9 is just one of many similar systems available to bacteria and archaea. Scientists divide them into two classes. The first class includes CRISPR systems of types I, III and IV, the second – II and V. Type II has the Cas9 protein involved in the acquisition of new spacers, the accumulation of SRNA in the cell and the cutting of DNA. In other systems, multi-protein complexes are used for these purposes. This makes Type II the simplest type of CRISPR systems suitable for the needs of genetic engineering.

Types can, in turn, be subdivided into subtypes depending on which additional genes are associated with CRISPR. Thus, IIA systems contain the csn2 gene, which encodes a protein that binds to DNA and participates in the acquisition of spacers. CSN2 is absent in IIB systems, but there is a cas4 gene, whose function is still unknown, and IIC systems have neither csn2 nor cas4.

All known CRISPR systems have been discovered by scientists in bacteria grown in laboratory conditions. However, there are a huge number of uncultivated microorganisms, which include both archaea and bacteria. They usually live in extreme conditions – mineral springs or toxic reservoirs in abandoned mines. However, researchers can isolate DNA from them and identify specific areas in it. In a new paper published in Nature on December 22, geneticists from the University of California at Berkeley decoded genomes from natural microbial communities, discovering other varieties of the CRISPR system.

Scientists have managed to find out that some species of little-studied archaea-like ARMAN nanoorganisms also possess CRISPR/Cas9, although previously it was believed that only bacteria have them. It is noted that this system occupies an intermediate place between subtypes IIC and IIB and can serve as a defense against parasitic "jumping" genes (transposons) entering the microorganism from other archaea. An attempt to reproduce the activity of archaeal CRISPR/Cas9 in Escherichia coli (Escherichia coli) did not lead to anything, which indicates the existence of some additional specific mechanisms regulating the system.

New types of systems within the second class – CRISPR/CasX and CRISPR/CasY - were also isolated from bacteria living in groundwater and sediments. The CRISPR/CasX system includes Cas1, Cas2, Cas4 and CasX proteins. The latter, as shown by experiments on E.coli, is distinguished by nuclease activity, that is, it is capable of splitting foreign DNA like Cas9. However, this happens only if there is a TTCN sequence in front of the protospacers, where N is any of the four nucleotides. Such sequences are called PAM (protospacer adjacent motif – motif adjacent to protospacer). The CRISPR/Cas9 system also has its own PAM – NGG, which should be located after the protospacer. In addition, CRISPR/CasY is able to cut DNA if there is a TA PAM sequence next to the target site.

What is the promise of this discovery? The fact is that the detected systems are the most compact known at the moment. According to scientists, the small amount of proteins necessary for their work makes CRISPR/CasX and CRISPR/CasY convenient tools for editing DNA. Moreover, metagenomic studies, in which DNA obtained from the environment is studied, will allow us to discover other varieties of CRISPR systems useful for genetic engineering.

Of course, there is an alternative to the search for CRISPR systems in nature - the improvement of the already existing CRISPR/Cas9 technology. Despite her high accuracy, she makes mistakes by cutting DNA in the wrong place. This makes it difficult to make the right changes to the genes and, therefore, effectively treat hereditary diseases. Therefore, scientists are looking for ways to improve the system. In 2016, many scientific papers were carried out on the modification of CRISPR and its transformation into various gene tools.

So, on December 8, the journal Cell published an article by scientists from the University of Toronto who created an "anti-CRISPR" system that turns off the mechanism under certain conditions. This allows you to suppress the activity of Cas9 if the guide RNA contacts the wrong fragment, and prevent errors. "Anti-CRISPR" consists of three nuclease inhibiting proteins. Naturally, it was originally invented not by scientists, but by viruses, which thus neutralized the immunity of bacteria.

In June, American researchers together with colleagues from Russia confirmed that the CRISPR/C2c2 system, derived from the fusobacterium Leptotrichia shahii, is capable of cutting single-stranded RNA. As a result, the CRISPR system can be used for knockout – suppression of functions – of matrix RNA, which transfers information from genes to ribosomes, where proteins are synthesized on its basis.

In May, specialists from the University of California created CRISPR-EZ technology, which allows inserting new DNA molecules into the genomes of mouse embryos with almost one hundred percent success. The CRISPR/Cas9 system is introduced into fertilized animal eggs using a microscopic needle and a tiny electric discharge. In their experiment, scientists managed to introduce a mutation into the gene in 88 percent of mice. This exceeds the number of genetically modified mice obtained using the CRISPR-editing method, which uses conventional injections.

In April, using a mutant variant of Cas9, which lacks nuclease activity, molecular biologists from the Medical School at the University of Massachusetts developed CRISPRainbow technology. The guide RNA indicating where to attach the enzymes contained fluorescent tags, which allows, for example, tracking the movement of mobile genetic elements.

Scientists are already using CRISPR systems to create genetically modified organisms, for example, malaria mosquitoes that spread harmful genes among their wild relatives. This method is called gene drive. If one of the parents of an individual was a carrier of a mutant gene, then he will pass it on to his offspring with a 50 percent probability. This happens because the parent has two copies of the gene, and only one of them is defective. CRISPR is able to copy a mutant fragment and insert it into a healthy gene. As a result, the descendants receive a mutation with a 100 percent probability.

In 2016, the experts of the Recombinant DNA Advisory Committee (RAC) approved the application of the University of Pennsylvania to conduct tests on human genetic modification using CRISPR/Cas9 technology.

However, they were overtaken by the Chinese. In November, the journal Nature reported that Chinese scientists introduced cells with genes modified by the CRISPR/Cas9 system into humans for the first time. The researchers extracted immune cells (T-lymphocytes) from the blood of a patient affected by metastatic lung cancer, and then used CRISPR technology to disable the gene encoding the PD-1 protein. The latter has been shown to suppress the immune system, promoting tumor growth. Scientists cultured the edited cells, increasing their number, and then injected them back into the human body. Whether gene therapy will cope with the disease is still unknown.

CRISPR systems can also be used to combat HIV and hereditary human diseases. However, further research is needed to develop effective treatments. Of course, we are not talking about mutants, as in science fiction films, but scientists will be able to quickly create genetically modified organisms, such as agricultural plants with resistance to parasites. It remains to be wondered why the scientists who discovered CRISPR and figured out how to use it have not yet received the Nobel Prize.

Portal "Eternal youth" http://vechnayamolodost.ru 23.12.2016

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