13 February 2014

Genomic surgery

Over the past decade, as DNA sequencing technology has improved and become cheaper, there has been a rapid deepening of a person into the secrets of his own genome. However, until recently, attempts by scientists to specifically modify genes in a living cell did not give the desired results. As an example, sickle cell anemia can be cited. This severe and often fatal disease is caused by a mutation of one of the three billion pairs of nucleotide bases of the patient's DNA. This genetic error is very simple and well studied, but despite this, researchers have long been powerless in trying to correct it and eliminate the harmful effects caused by it.

However, recent advances in genomic engineering are encouraging. One of the most promising is CRISPR technology. It allows performing microsurgical operations on genes by changing the DNA sequence in strictly defined regions of the chromosome. Along with the TALENs technology invented a few years ago, as well as its predecessor using protein complexes known as "zinc fingers", CRISPR can expand the scope of gene therapy and provide the possibility of curing simple genetic diseases such as sickle cell anemia, and in the future – more complex pathologies affecting several genes at once. Most traditional gene therapy approaches consist in embedding new genetic material into the genome of a cell randomly and only allow you to add a whole gene. CRISPR, as well as other new technologies, on the contrary, give scientists the opportunity to edit specific DNA fragments with high accuracy, up to the replacement of one pair of nucleotide bases. In fact, they allow you to rewrite the human genome at will.

Of course, more than one year will pass before the introduction of these approaches into clinical practice, but researchers have already achieved some success in preliminary experiments devoted to the development of methods for the treatment of sickle cell anemia, HIV and cystic fibrosis (Table 1). One of them, Gang Bao from the Georgia Institute of Technology, successfully used CRISPR to correct a mutation that causes sickle cell anemia in the DNA of cultured human cells. His group started working with the "zinc fingers" technology in 2008, after which it switched to TALENs, and subsequently to CRISPR. Bao hopes to expand the scope of his work to other diseases in the future, however, he believes that sickle cell anemia is an ideal object for testing the approach, since this disease is caused by a single nucleotide mutation in one gene and affects cells of the same type.

To date, the only effective method of treating sickle cell anemia is bone marrow transplantation, which is possible only if the patient has a healthy compatible donor. However, even in this case, there is a high risk of death of the patient from developing immunological reactions and other side effects.

The approach developed by Bao will help to avoid such troubles. It involves isolating hematopoietic stem cells from the patient's bone marrow, correcting their genome using CRISPR and injecting them back to the patient. Such "corrected" cells will divide and differentiate into normal red blood cells. Even if in this way it is possible to replace 50% of the red blood cells affected by the disease, the patient will feel much better, and replacing 70% of the abnormal cells will completely cure him.

Despite the fact that CRISPR technology is only a little more than a year old, it has already transformed the direction of genetic research. Scientists were able to quickly and simultaneously make several changes to the genome of living cells. Multiple variants of both pathologically altered and normal genes are involved in the development of many human diseases, including heart disease, diabetes mellitus and various neurological pathologies. Studying these complex interactions in animal models is a very slow and inefficient process. When using traditional genetic methods, it can take a year to create a genetically modified mouse model with one mutation. If researchers need an animal model with several mutations, genetic changes have to be made sequentially, which stretches the experiment for several years. CRISPR allows you to create such models in a few weeks.

Table 1.Correction methods


Nuclease complexes "zinc fingers"




What is it?

A protein complex consisting of a DNA-cutting nuclease enzyme and a DNA-binding region that recognizes the target gene.

There is also a protein complex consisting of a DNA-cutting nuclease enzyme and a DNA-binding region that recognizes the target gene, but its synthesis is much simpler than the synthesis of the zinc fingers complex.

A DNA-cutting enzyme guided by an RNA molecule selectively binding to the target gene.

Advantages and disadvantages

This technology was the first programmable tool for genome correction, but it involves the use of proteins that are difficult to modify for use with new target genes. Potentially dangerous unintended DNA incisions may also occur.

Despite the relative ease and cheapness of synthesis compared to zinc finger complexes, TALENs proteins are quite difficult to synthesize and deliver inside cells. Also a problem is the possibility of unintentional DNA cuts.

This technology is accessible and easy to use. It allows for high-performance experiments on simultaneous correction of several genes. Just as with other methods, there is a risk of unintentional DNA incisions.

CRISPR-RNAs, which with high accuracy guide the enzyme nuclease to the site of the intended DNA cutting, can be easily synthesized for any target gene. In December 2013, researchers at the Massachusetts Institute of Technology, working under the leadership of Feng Zhang and Eric Lander, created a library of existing CRISPR RNAs, each of which is specific to a specific human gene. This extensive collection, affecting almost all human genes, is available to other interested researchers, which will speed up genome-wide research into the genetics of cancer and other diseases.

Genomic GPSGenetic engineering originated in 1973 when Herbert Boyer and Stanley Cohen embedded altered foreign DNA into the genome of a bacterium.

Within a few years, Genentech was founded, which began using genetically modified E. coli to produce human insulin intended for the replacement therapy of diabetes mellitus. In 1974, Rudolph Jaenisch created the first transgenic mouse using a virus that embedded a DNA fragment of another species into the animal's genome. However, in these and other examples of early genetic engineering, the researchers' capabilities were limited by techniques that embed foreign DNA into the genome of the target cell randomly. That is, scientists could only rely on luck.

It took molecular biologists more than twenty years to learn how to effectively change certain genes in animal DNA. Dana Carroll from the University of Utah suggested that protein complexes containing the enzyme nuclease, called "zinc fingers", synthesized in 1996 by Johns Hopkins University researchers, can be used as a programmable tool for gene modification. One of the ends of such a complex recognizes a certain DNA sequence, and the other cuts it. The resulting incision is subsequently restored using a fragment of foreign DNA. This technology allowed scientists to carry out targeted correction of chromosomes, but it turned out to be very difficult in practical application. Each modification requires the creation of a new protein specific to the target sequence. This task requires a lot of time and effort, while capricious protein complexes do not always work.

The next breakthrough in the field of gene editing occurred in 2010 with the advent of TALENs technology. This approach also involves the use of proteins that find and cut a specific DNA sequence. However, their modification to target a specific sequence of nucleotides is much simpler. Despite the significant advantages over "zinc fingers", the proteins used by TALENs technology have very large molecules that are difficult to work with, including introducing them into the cell.

The advent of CRISPR has completely changed the situation. In this technology, DNA-specific proteins are replaced by short RNA fragments, the 20-nucleotide sequences of which are easily synthesized in the laboratory.

CRISPR stands for "clustered regularly interspaced short palindromic repeats" – short palindromic repeats regularly arranged in groups. These repeats are often found in bacterial genomes, short DNA sequences that are equally readable in both directions. For the first time, these repeats were identified in the 1980s, but it was only almost 20 years later that researchers established that they were a component of the bacterial cell protection system. In a viral attack, a bacterium can embed viral DNA in its genome between repeatedly repeating segments. Upon subsequent encounter with the same virus, the DNA of these regions is used to synthesize RNA chains that recognize the corresponding sequences of viral DNA. The enzyme attached to these RNAs cuts the viral DNA.

In 2012, Emmanuelle Charpentier from the Center for the Study of Infectious Diseases named after Helmholtz and Jennifer Doudna from the University of California at Berkeley demonstrated the possibility of using RNA in combination with the Cas9 enzyme for targeted cutting of DNA in solution. In 2013, Feng Zhang and George Church from Harvard University independently published results proving that the CRISPR/Cas9 system can be used to modify the genome in mammalian cells, including humans.

Currently, researchers wishing to modify a gene only need to synthesize the Cas9 enzyme and an RNA fragment corresponding to the sequence of the desired region of the target protein. The RNA directs the enzyme to the planned place of cutting the DNA molecule. Since the same enzyme is used independently of the target, researchers can plan experiments on simultaneous correction of several genes using Cas9 and the required number of guide RNAs.

Complex riddlesFeng Zhang, in addition to the Massachusetts Institute of Technology, who is an employee of the Broad Institute and the McGavern Institute for Brain Research, is interested in the genetic background of mental illness.

To understand the pathogenesis of these complex pathologies, he participated in the development of many tools for the modification of genes and neurons, including TALENs and optogenetics, a technology for regulating the activity of neurons using laser light. As soon as information about the appearance of CRISPR reached him in 2011, he began to modify this technique for use on human cells. Currently, Chzan uses it to study genetic secrets that hide the causes of such severe and poorly understood diseases as schizophrenia and autism.

The CRISPR technique allows Chzan to systematically test DNA variants considered to be associated with these diseases. Despite the fact that significant progress has been made over the past decade in identifying genes that are often found in people with mental illness, scientists are unable to understand how these genes are interconnected with their symptoms. According to Chzan, the data obtained as a result of sequencing is essentially just a record of observations. In order to understand whether a suspected gene really causes a particular symptom, it is necessary to introduce an appropriate mutation into a healthy organism and observe the consequences. The appearance of signs of disease in such a cell or organism is proof of the involvement of the gene under study.

Table 2.


The Path to Healing Sickle cell anemia



Cystic fibrosis


Correction of the mutation causing sickle cell anemia in stem cells isolated from the patient's bone marrow and their subsequent introduction back to the patient; alternatively, the fetal hemoglobin gene in the same cells can be reactivated in the inactivated state.

Prevention of infection of healthy immune cells of HIV-infected patients by changing the genes of progenitor cells used by the virus to penetrate mature cells. Alternatively, inactive HIV particles embedded in the human genome can be destroyed by altering important viral genes.

Correction of cystic fibrosis-causing mutations in the epithelial cells of the respiratory tract and other tissues suffering from the disease cells.


The correction strategy works effectively on human cells in the laboratory; "zinc fingers", TALENs and CRISPR have been successfully used for this. The effectiveness of the reactivation strategy has been demonstrated on human cells and mice using "zinc fingers".

A preventive strategy using "zinc fingers" is currently undergoing clinical trials; the effectiveness of using TALENs and CRISPR has been demonstrated in laboratory conditions. The strategy of eliminating latent viral particles with the help of "zinc fingers" and CRISPR also worked in experiments on cells.

Zinc Fingers and TALENs have been successfully used to correct cystic fibrosis-causing mutations in respiratory tract cell cultures; CRISPR has been used to correct mutations in cultured organ-like structures grown from intestinal cells.

Chzan can recreate genetic variants found in people with autism and schizophrenia, in cultured human cells and in cells of live laboratory mice. This allows you to create mice with a human mutation in the corresponding gene and analyze the features of their behavior and learning ability. After that, it is possible to evaluate the behavior and physiology of neurons cultured in the laboratory grown from stem cells modified by the same mutation.

Zhang also uses CRISPR to make changes to multiple genes at the same time. This possibility is especially important when studying such complex diseases as autism and schizophrenia, which, unlike sickle cell anemia, are not the result of a single known mutation. In different patients, diseases develop due to different combinations of mutations. Solving this extremely complex puzzle requires large-scale systematic research devoted to the study of the effects exerted by various genes and the mechanisms of their interaction. CRISPR makes this task doable.

Children on an individual projectAt the end of last year, Dudna, Chzan, Church and two other pioneers in the field of genomic engineering founded the startup company Editas Medicine, whose activities are devoted to the development of new methods of treating human genetic diseases.

In November, the company announced that it had raised $43 million in venture capital and unveiled plans to use genome correction technologies to treat a wide range of diseases.

The appearance of Editas Medicine caused a great resonance due to the revival of interest in gene therapy caused by the achievements of recent years, including the development of safe mechanisms for the delivery of genetic material to cells. The treatment methods developed by the company will be fundamentally different from earlier approaches involving the use of carrier viruses to deliver therapeutic genes.

According to Church, correcting an existing gene or removing a fragment of it is beyond the capabilities of methods based on the use of viral vectors. At the same time, removing a tiny piece of DNA can be much more effective than embedding a healthy gene. As an example, Huntington's disease can be cited. This fatal brain disease develops as a result of the accumulation of toxic protein aggregates in neurons. The introduction of a normal copy of the gene into the genome of cells does not eliminate the toxic protein synthesized by cells. You can get rid of it only by "rewriting" the original version of the gene.

CRISPR's capabilities are not limited to adding the missing and correcting errors. Church adds that when you begin to realize that the most common versions of genes are not necessarily optimal, a much more extensive field of activity opens before your eyes. Perhaps in the future, specialists will be able to modify normal genes in such a way that a person will be able to resist infectious diseases more effectively. Theoretically, it is even possible to make corrections to the molecular mechanisms of the aging process.

Church also predicts that if genome correction methods are used to treat childhood diseases, specialists will be tempted to use them to engineer embryos during in vitro fertilization. The effectiveness of this approach has already been demonstrated in experiments on mouse and rat embryos. And at the end of January, Chinese scientists announced the creation of genetically modified monkeys using CRISPR.

These opportunities cannot but raise ethical questions. However, if researchers can prove the safety of getting rid of diseases by correcting the genome, some parents will inevitably have a desire to interfere with the genomes of healthy embryos. If gene therapy makes it possible to prevent mental retardation, questions will invariably arise about the possibility of making a whole range of intellectual improvements.

As the field of practical application of CRISPR expands, the emergence of such questions is inevitable. However, today this technology is under development. While researchers, including Bao, Church and Zhang, dream of getting rid of incurable diseases, in reality they are mainly engaged in improving technology and studying its capabilities. However, already now, shortly after its appearance, CRISPR technology has completely transformed the specialists' view of the possibilities of genome correction and endowed them with truly unlimited possibilities.

Evgeniya Ryabtseva
Portal "Eternal youth" http://vechnayamolodost.ru based on the materials of MIT Technology Review: Genome Surgery.


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