Scientists have high hopes for the CRISPR / Cas9 technology, which allows high-precision changes in the genomes of living organisms, including humans. All new scientific articles are published describing various types of CRISPR systems, as well as their modifications. "Lenta.ru" talks about the discoveries in this area, made in 2016.
Thousands of them
CRISPR / Cas9 is a bacterial adaptive immunity system that allows microorganisms to fight viruses. It consists of spacers - DNA sections corresponding to certain fragments (protospacers) of the infectious agent's DNA. Spacers encode specific crRNA molecules that bind to the Cas9 enzyme. The resulting complex attaches to the virus's DNA chain, and Cas9 works like a pair of scissors, cutting it.
In fact, CRISPR / Cas9 is just one of many similar systems that bacteria and archaea have. Scientists divide them into two classes. The first class includes CRISPR systems of types I, III, and IV, and the second - II and V. Type II contains the Cas9 protein involved in the acquisition of new spacers, the accumulation of crRNA in the cell, and DNA cleavage. In other systems, multi-protein complexes are used for these purposes. This makes type II the simplest type of CRISPR system suitable for genetic engineering needs.
Types can, in turn, be subdivided into subtypes depending on which additional genes are associated with CRISPR. For example, IIA systems contain the csn2 gene, which encodes a protein that binds to DNA and is involved in the acquisition of spacers. In IIB systems, csn2 is absent, but there is a cas4 gene, whose function is still unknown, and IIC systems have neither csn2 nor cas4.
Treasures inside bacteria
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All known CRISPR systems have been discovered by scientists in bacteria grown under 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 bodies of water in abandoned mines. However, researchers can isolate DNA from them and identify specific regions in it. In a new paper published in Nature on Dec. 22, geneticists at the University of California, Berkeley have decoded genomes from natural microbial communities, discovering other variations of the CRISPR system.
Bacteriophage - a virus that infects bacteria
Image: Giovanni Cancemi / Depositphotos
Scientists managed to find out that some types of little-studied arche-like nanoorganisms ARMAN also possess CRISPR / Cas9, although it was previously 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 E. 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 identified from bacteria living in groundwater and sediments. The CRISPR / CasX system includes proteins Cas1, Cas2, Cas4 and CasX. The latter, as shown by experiments on E. coli, is distinguished by nuclease activity, that is, it is capable of cleaving foreign DNA like Cas9. However, this happens only if the TTCN sequence is in front of the protospacers, where N is any of the four nucleotides. Such sequences are called PAM (protospacer adjacent motif - motif adjacent to the 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 near the target site.
What is the promise of this discovery? The fact is that the detected systems are the most compact ones known at the moment. According to scientists, the small amount of proteins required for their work makes CRISPR / CasX and CRISPR / CasY convenient tools for DNA editing. Moreover, metagenomic research, in which DNA obtained from the environment is studied, will reveal other types of CRISPR systems that are useful for genetic engineering.
The path to excellence
Of course, there is an alternative to searching for CRISPR systems in nature - improving 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 correct changes in genes and therefore effectively treat hereditary diseases. Therefore, scientists are looking for ways to improve the performance of the system. In 2016, there have been many scientific papers devoted to modifying CRISPR and turning it into various gene tools.
Protein Cas9 and crRNA - the main components of the CRISPR system
Image: Steve Dixon / Feng Zhang / MIT
So, on December 8, the journal Cell published an article by scientists from the University of Toronto, who created "anti-CRISPR" - a system that turns off the mechanism under certain conditions. This makes it possible to suppress Cas9 activity if the guide RNA binds to the wrong fragment and prevents errors. Anti-CRISPR is composed of three nuclease inhibiting proteins. Naturally, initially it was not invented 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, obtained from the fusobacterium Leptotrichia shahii, is capable of cutting single-stranded RNA. As a result, the CRISPR system can be used to knockout - suppress functions - of messenger RNA, which carries information from genes to ribosomes, where proteins are synthesized on its basis.
In May, researchers at the University of California created CRISPR-EZ, a technology that allows new DNA molecules to be inserted into the genomes of mouse embryos with almost 100% success. The CRISPR / Cas9 system is introduced into fertilized animal eggs using a microscopic needle and a tiny electrical discharge. In their experiment, scientists managed to introduce a mutation in the gene in 88 percent of the mice. This exceeds the number of genetically modified mice produced by CRISPR editing, which uses conventional injections.
In April, using a mutant variant of Cas9 that lacks nuclease activity, molecular biologists at the University of Massachusetts School of Medicine developed the CRISPRainbow technology. The guide RNA, which indicates where the enzymes bind, contained fluorescent labels, which makes it possible, for example, to track the movement of mobile genetic elements.
Anopheles mosquito may be the first victim of CRISPR systems
Photo: Jim Gathany / Wikipedia
Brave new world
Scientists are already using CRISPR systems to create genetically modified organisms, such as the malaria mosquito, 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 is 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 offspring get a mutation with 100% probability.
In 2016, experts from the Recombinant DNA Advisory Committee (RAC) approved an application from 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 for the first time introduced cells with genes modified by the CRISPR / Cas9 system into humans. The researchers extracted immune cells (T lymphocytes) from the blood of a patient with metastatic lung cancer and then used CRISPR technology to turn off the gene that encodes the PD-1 protein. The latter has been shown to suppress immunity by promoting tumor growth. Scientists cultivated the edited cells, increasing their number, and then injecting them back into the human body. Whether gene therapy will cope with the disease remains to be seen.
CRISPR systems can also be used to combat HIV and human hereditary diseases. However, further research is required 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 seen why the scientists who discovered CRISPR and figured out how it could be used have not yet received the Nobel Prize.
Alexander Enikeev