16 December 2019

CRISPR-Cas9 and its features

A crack in the universe


Corpus Publishing House presents the book by Jennifer Dudna and Samuel Sternberg "A Crack in the Universe. Genome editing: an incredible technology capable of controlling evolution" (translated by Svetlana Yastrebova).

Jennifer Doudna, an American biochemist, professor at the University of California at Berkeley, is one of the creators of the CRISPR–Cas9 genome editing technology. A method that allows you to precisely change the DNA of a living organism can completely change the world of medicine in the coming years.  However, the possibilities of CRISPR-Cas9 are infinitely wider: for example, with the help of this technology, it will be possible to create hybrid animals or "edit" the appearance of an unborn child during artificial insemination. Jennifer Dudna talks about the development of the CRISPR-Cas9 method, describes the prospects for its application and shares the concerns associated with it.

The proposed passage deals with the use of genome editing in medical experiments on animals and the development of xenotransplantation.


Animal experiments are vital for studying human diseases, whether they are carried out to confirm the genetic causes of specific ailments, to test potential drugs or to evaluate the effectiveness of medical intervention, whether surgical methods or cell therapy. The necessary starting point here is a good genetic model, that is, an animal that, in its physical and genetic parameters and condition, is as close as possible to what is observed in this group of patients. CRISPR offers an effective and simple approach here.

Since the beginning of the XX century, the main model organism for biological and medical research has been the house mouse (Mus musculus), in which 80% of the genes coincide with human ones. In addition to the fact that mice are our close genetic relatives, they have other obvious advantages. Mice and humans have similar physiology of various systems – immune, nervous, cardiovascular, musculoskeletal and others. Mice can be bred in captivity, it is not difficult and inexpensive to keep them, since they are small, peaceful and prolific animals. The "accelerated" course of their life – one year of mouse life is approximately equal to thirty human – means that their entire life cycle can be observed in the laboratory in just a few years. And, perhaps most importantly, with the help of various methods – of which CRISPR is the most modern and the most powerful – genetic manipulations can be carried out on mice to simulate a variety of human diseases and conditions. Millions of mice are bred and supplied to researchers around the world every year, and there are over thirty thousand unique lines of these rodents used to study a wide variety of diseases – from cancer and heart disease to blindness and osteoporosis.

However, the use of mice as model organisms has some limitations, because in the case of many human ailments – for example, cystic fibrosis, Parkinson's and Alzheimer's diseases, Huntington's chorea – these animals do not show the main symptoms or react to potential treatments atypically. As a result, there is a gap in the sequence "from study to patient" (bench-to-bedside) – that is, in the process during which the results of scientific work in the laboratory become the basis for new methods of treatment in the clinic.

CRISPR will be able to fill this gap by making disease modeling in other animals almost as accessible as in mice. Progress can already be seen today on the example of non-human primates. Transgenic monkeys were first created in the early 2000s, when researchers used viruses to inject foreign genes into animal genomes, but there were no monkeys with an edited genome before the advent of CRISPR. Only at the beginning of 2014, a team of Chinese scientists created Javanese macaques with an edited genome by introducing CRISPR into embryos at the single cell stage – a similar method was tested on mice a year earlier. In this study, scientists programmed CRISPR in such a way that its targets were two genes at the same time: one associated with severe combined immunodeficiency syndrome (TCID) in humans, and the other is associated with obesity (both of them obviously affect our health). Later, other scientists created Javanese macaques with an altered gene, mutations in which are observed in about 50% of human cancers, as well as rhesus macaques with mutations that cause Duchenne myodystrophy. Genome editing is also used to work with genes associated with diseases of the nervous system, while monkeys have a unique advantage as model organisms: only they can be used to study behavioral and cognitive abnormalities found in humans.

In a way, I feel uncomfortable thinking about using monkeys like this, but I also realize how important it is to develop methods to eliminate symptoms and treat human diseases to alleviate our suffering. Monkeys with an edited genome can serve as a reliable replacement for human patients, allowing scientists to look for ways to cure without putting human lives at risk.

Pigs, thanks to CRISPR, have also become popular animals for modeling human diseases. The anatomy of pigs is close to human, these animals grow quickly, and they have a large litter size. I believe that, subject to the development of good methodological guidelines, the use of farm animals for biomedical research is more acceptable than the use of companion animals ("pets"), such as primates. In fact, pigs with an edited genome have already been used as model organisms to study pigmentation defects, various variants of hearing loss, Parkinson's disease and immunological disorders, and this list continues to grow.

Some scientists also consider pigs as a potential source of medicines. In the near future, we may be using pigs as bioreactors to produce valuable drugs, for example, therapeutic human proteins, which are too complex to synthesize from scratch and can only be produced in living cells. Scientists have already been thinking about what other transgenic animals can produce such biopharmaceuticals.

The first such drug approved by the FDA is an anticoagulant called antithrombin, and it is obtained from the milk of genetically modified goats. Another drug approved by the agency is isolated from the milk of transgenic rabbits, and in 2015 the FDA gave the green light to a protein drug extracted by purification of proteins from eggs of transgenic chickens. Obtaining such drugs from transgenic animals, rather than from cell cultures, has a number of advantages: a larger amount of product, easier scaling of production and lower costs. CRISPR guarantees further improvement of pharmaceutical production, primarily providing scientists with much better control over the creation of transgenic animals.

For example, experiments on pigs have shown that CRISPR allows unmistakably replacing pig genes with human ones, which makes it possible to more effectively isolate therapeutic proteins encoded by these genes. If we take into account that many of the world's best-selling pharmaceuticals are based on proteins, then the huge potential of using genome editing in this field of medicine becomes obvious.

Some scientists hope that pigs can provide medicine with even more: an abundant renewable source of whole organs for xenotransplantation. This is not a new idea; scientists have been considering pigs as candidates for such a source for a long time for the same reasons that they are used to model diseases – they are easy to breed, they multiply quickly, and their organs are extremely close to human in size.

However, this dream is unlikely to be realized in the near future. There is a whole array of protective immune mechanisms working in our body, and therefore the rejection of donor organs is a serious problem for both doctors and patients, even when it comes to transplantation from person to person. So far, there are literally a few cases of xenotransplantation, when the transplanted organ managed to be kept in the recipient's body for at least some time.

At the same time, the world has never been in such dire need of new methods of transplantation as it is now. In the United States alone, over 124,000 patients are currently on the waiting list for this procedure, while only about 28,000 transplants are performed per year. According to some estimates, one more person is added to this queue every ten minutes and on average twenty–two people a day die waiting for their transplants - or their condition worsens so much that they will no longer be able to undergo transplantation. The main reason for this catastrophic situation is the lack of donor organs.

New technologies, including CRISPR, make it possible to create pigs with organs suitable for human transplantation. Previous achievements in this field have mainly been associated with the transfer of human genes into the pig genome, so that its organs are not subjected to too severe rejection, which threatens any xenograft. Now genome editing is used to turn off those pig genes that can provoke an immune response in humans, and eliminate the risk of human infection with viruses embedded in the pig genome. Finally, thanks to cloning technologies, it has become possible to organically combine various genetic changes in one animal. The head of a well–known company, whose activities are related to this area, outlined his main goal as follows: "to provide an unlimited supply of organs suitable for transplantation" - that is, those that can be made to order.

This project is still at the very early stage, but the use of humanized GM pigs has already helped to achieve a number of outstanding achievements: a pig kidney transplanted to a baboon has worked for over six months, and a pig heart in a baboon's body has successfully taken root for two and a half years. Tens of millions of dollars have been allocated for future research in this area, and a company called Revivicor has already announced that it intends to raise a thousand pigs a year in laboratories with state-of-the-art operating rooms and helicopter pads, so that fresh organs can be quickly delivered whenever they are needed. The start of clinical trials of xenotransplantation seems only a matter of time – once CRISPR technology opens a new path for patients in dire need of new organs and new drugs.

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