The invention of the CRISPR-Cas gene editor is called a revolution in biology. Scientists promise to use it to improve plant varieties and animal breeds for agriculture, to treat congenital genetic diseases in humans. The RIA Novosti correspondent went to see who and how the genome is being edited.
The Genome Editing Center of Moscow State University opened a little over a year ago - on the campus. I am met by its director, Doctor of Chemistry Peter Sergiev, and while we are riding in the elevator, he devotes a little to the history of genetic engineering.
“The genome has been edited before. But it was difficult to create tools for this, it took a long time, and the result was often not the way we wanted it,”he says.
In the vivarium, Pyotr Vladimirovich changes into blue laboratory clothes, they give me a disposable white overalls and ask me to wipe the camera with alcohol. Now Petr Vladimirovich looks like a surgeon, and I - like a laboratory assistant from the series "CSI - Crime Scene".
In the sterile laboratory, a container with fertilized mouse eggs and a test tube with RNA solution have been prepared for us. That's all you need for CRISPR-Cas genome editing.
CRISPR is an English acronym for a phrase that literally means "regularly grouped short palindromic repetitions." In fact, these are just small sections of the DNA of bacteriophage viruses embedded in the bacterial genome. These sequences are needed for the bacterial immune system to function and serve as a sort of "police wanted" announcement. If the bacterium survives after being infected with a bacteriophage, it uses the Cas protein enzymes to cut a piece of its DNA and insert it into its genome to be recognized later. The bacterial genome inherits this library of "enemies" carefully collected by previous generations.
In 2013, scientists found out that the Cas protein works in any organism, including mammals. He is able to make directional breaks in both strands of the DNA molecule and thus change the genome.
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Egg manipulation
Petr Sergiev makes a glass capillary with a diameter of one hundred microns at the micro forge, draws the egg into it and transfers it under glass with a nutrient medium. The finished sample is sent to the stage of the optical microscope. The monitor displays an enlarged image of the egg, quivering at the tip of the capillary. Two rounded spots stand out - these are pronuclei with the DNA of the mother and father.
The scientist prepares another capillary with an order of magnitude smaller in diameter. This is an injection syringe. It collects a solution with two types of RNA into it and, with the help of manipulators, gently pierces the egg. That's it, the injection is done.
RNA injected into the egg is needed to cut the genome at a given location. One type of RNA contains a strictly defined sequence of nucleotides, similar to the one we intended to change. The task of this RNA is to find the corresponding region in DNA, therefore it is called "guide". RNA of the second type, matrix, is a kind of instruction for the synthesis of the Cas9 nuclease protein. This protein acts as a catalyst for a chemical reaction that breaks phosphodiester bonds between well-defined DNA bases. Since the protein molecule has two nuclease centers, both DNA strands are opened, moreover, in the place, the coordinates of which Cas9 "counted" with the guide RNA.
The DNA molecule perceives the break in both strands as a serious breakdown and seeks to repair it. Exonuclease enzymes floating in the cell immediately remove several nucleotides from both ends of the break. This is enough to break, or, as geneticists say, “turn off” a gene.
How the CRISPR-Cas9 genomic editor works / Illustration by RIA Novosti. Alina Polyanina.
If a directed mutation is needed, for example, it is required to mark a protein in order to track it in the body, then another template is added to the complex to two RNAs in the form of a specially designed DNA. It consists of sequences identical to the edges of the future break, and also contains the section to be inserted. After Cas9 has cut the DNA molecule, its ends are connected using this additional template, so the set of nucleotides we need is inserted into the break.
“We can attach a small tail to the squirrel, by which it is convenient to pull it out and see what it interacts with. We can insert a gene, but it is not as effective as turning it off,”the scientist continues to explain.
GMOs and chimeras
The edited egg will be transplanted into the uterine tube of a surrogate mouse. She lived for some time with a male whose seminiferous tubules were tied. The couple had a normal sex life, but could not conceive. Nevertheless, the mouse's body believed that she was pregnant, and formed the appropriate hormonal background for her. Now this female is bearing someone else's fetus.
In three weeks she will give birth to the most ordinary mice. Scientists will wait until the rodents grow up, take a small piece of their tail from them and use PCR to analyze the piece of DNA that they edited. A mutation or a disabled gene is found in more than half of the cases. The reverse procedure - insertion of a sequence into the genome - succeeds in no more than 10% of experiments.
Various interesting effects appear when editing. For example, mosaic mice, or chimeras, are born, which have cells with different variations of the maternal and paternal genomes. Cas9 can cut DNA many times, but the messenger RNA encoding it is not eternal, and the injected solution simply disappears in a series of cell divisions. Sometimes the editor still fires again after the pronuclei have fused and the egg has shared. And since DNA repair after a rupture is always a random process and healing never happens in the same way, then some of the cells in one organism will contain another mutation.
For science and medicine
We move into the adjacent room to watch the live results of the genome-editing experiments. On the shelves on the left - containers with genetically modified mice, on the right - with common rodents for control. They were grown, like those on the left, but the genome was not manipulated. Control mice are needed in order to have a norm in front of our eyes and compare the creatures obtained in the experiment with it.
Pyotr Sergiev takes one of the containers with a pair of gray mice. Outwardly, they are completely ordinary, but they do not have offspring. The fact is that in the male the gene of one of the RNA methyltransferases, an enzyme produced only in sperm, is turned off. Males with an inactive gene are born sterile. The exact purpose of the gene and enzyme is still unknown. To find out, two strains of mice were bred in the laboratory: one had a gene turned off, the other had a protein marked with a genomic editor.
“A mutation in this gene is also found in humans - then a man suffers from infertility. But until we find out why it is needed, why it modifies RNA, we will not be able to help such patients,”the scientist reasons.
In fact, we still don't know the function of most human genes. Finding out this is a fundamental task solved by many research groups around the world, including in Russia. The mouse genome is very similar to that of humans. It is hoped that with the help of CRISPR-Cas, the study of the genome of any creature will go faster.
Sergiev's group together with N. N. NN Petrova set about looking for mutations leading to some types of cancer. The nearest plans include a project to create genetically modified animals for agriculture in cooperation with the All-Russian Research Institute of Animal Husbandry and the Institute of Gene Biology of the Russian Academy of Sciences.
“CRISPR-Cas is a fantastic tool that allows you to change the genome at your discretion, to be a little bit of God. Ultimately, the task of science is to understand how such a complex creature like a mammal works,”says the scientist.
Solving fundamental problems of science is great, but what will this technology give to medicine? In the foreseeable future, alas, not much. It's easy to edit an egg and raise a genetically modified mouse, but it's impossible to correct the genes of an adult animal, let alone a human.
Methods for editing DNA in somatic (that is, already formed cells) are still very ineffective. In order to introduce into a cell some gene turned off by mutation and force it to produce a certain protein, you need to remove some of the cells from the body, edit the DNA in them and put it back into the body. In principle, there is hope that in this way it will be possible to fight diseases such as Duchenne muscular dystrophy or cystic fibrosis, when it is necessary to restore the working capacity of some part of the cells. As for the predisposition to cancer, so far the genomic editor is powerless. If a mutant gene is found in a person, then it is found in all cells of the body. It is unrealistic to change all of them. And every cell with a mutation is a source of danger.
But even if CRISPR / Cas will help answer only some fundamental questions and allow the treatment of rare genetic diseases, it will still be a big step forward for humanity.
Tatiana Pichugina