Human Editing: How and Why Scientists Perform Genome Operations

Application of genetic engineering in scientific research

  • Gene knockout

To study the function of this or that

gene can be applied gene knockout. This is the name of the technique of removing one or more genes, which allows you to study the consequences of such a mutation.

For a knockout, the same gene or a fragment thereof is synthesized so that the gene product loses its function. The main methods of implementation: zinc finger, morpholino and TALEN.

To obtain knockout mice, the obtainedThe genetically engineered construct is introduced into embryonic stem cells, where the construct undergoes somatic recombination and replaces the normal gene, and the altered cells are implanted into the surrogate mother's blastocyst. In the fruit fly, Drosophila mutations are initiated in a large population, in which offspring with the desired mutation are then sought. Plants and microorganisms are knocked out in a similar way.

  • Artificial Expression

A logical addition to knockout isartificial expression, that is, the addition of a gene to the body that it did not have before. This genetic engineering technique can also be used to study the function of genes. In essence, the process of introducing additional genes is the same as for knockout, but existing genes are not replaced or damaged.

  • Gene product visualization

Used when the task is to studylocalization of the gene product. One of the methods of labeling is the replacement of a normal gene with a fusion with a reporter element, for example, with the gene of the green fluorescent protein GFP. This protein, which fluoresces in blue light, is used to visualize the product of gene modification.

While this technique is convenient and useful, its side effectsthe consequences may be partial or complete loss of function of the protein under study. A more sophisticated, albeit not so convenient, method is to add to the protein under study not so large oligopeptides that can be detected using specific antibodies.

  • Investigation of the Expression Mechanism

In such experiments, the task is to studyconditions of gene expression. Expression features depend primarily on a small piece of DNA located in front of the coding region, which is called a promoter and serves to bind transcription factors.

This site is introduced into the body by placing afterinstead of its own reporter gene, for example, GFP or an enzyme that catalyzes an easily detectable reaction. In addition to the fact that the functioning of the promoter in certain tissues at one time or another becomes clearly noticeable, such experiments make it possible to study the structure of the promoter by removing or adding DNA fragments to it, as well as to artificially enhance its functions.

Why is human genetic engineering needed?

When applied to humans, genetic engineering couldused to treat hereditary diseases. However, technically, there is a significant difference between treating the patient himself and modifying the genome of his offspring.

The task of changing the genome of an adultsomewhat more difficult than breeding new genetically engineered animal breeds, since in this case it is required to change the genome of numerous cells of an already formed organism, and not just one egg-embryo. For this, it is proposed to use viral particles as a vector.

Viral particles are able to penetratea significant percentage of the cells of an adult, embedding their hereditary information into them; possible controlled multiplication of viral particles in the body. At the same time, in order to reduce side effects, scientists try to avoid the introduction of genetically engineered DNA into the cells of the genital organs, thereby avoiding exposure to the patient's future descendants.

It is also worth noting the significant criticism of this technology in the media: the development of genetically engineered viruses is perceived by many as a threat to all mankind.

With the help of gene therapy, it is possible to change the human genome in the future. Currently, effective methods for modifying the human genome are under development and testing in primates.

Genetic engineering of monkeys for a long timefaced serious difficulties, but in 2009 the experiments were crowned with success: a publication appeared in the journal Nature about the successful use of genetically engineered viral vectors to cure an adult male monkey from color blindness. In the same year, the first genetically modified primate (grown from a modified egg), the common marmoset (Callithrix jacchus).

Although on a small scale, genetic engineering is alreadyused to give women with some types of infertility a chance to get pregnant. To do this, use the eggs of a healthy woman. As a result, the child inherits the genotype from one father and two mothers.

However, the possibility of introducing more significantchanges in the human genome is faced with a number of serious ethical problems. In 2016, a group of scientists in the United States received approval for clinical trials of a cancer treatment method using the patient's own immune cells, which are genetically modified using CRISPR / Cas9 technology.

At the end of 2018, two children were born in China,whose genome was artificially altered (the CCR5 gene was turned off) at the embryonic stage by the CRISPR / Cas9 method, as part of research conducted since 2016 to combat HIV. One of the parents (father) was HIV-positive, and the children, according to the statement, were born healthy.

Since the experiment was unauthorized (beforeAll such experiments on the human embryo were allowed only in the early stages of development with the subsequent destruction of experimental material, that is, without implanting the embryo into the uterus and giving birth to children), the scientist responsible for it did not provide evidence for his statements, which were made at an international conference on genome editing ...

At the end of January 2019, the Chinese authorities officially confirmed the facts of this experiment. In the meantime, the scientist was forbidden to engage in scientific activities and he was arrested.

How is the human genome edited?

  • Zinc fingers method

"Zinc fingers" are also found in the compositionhuman proteins. Thanks to this method, it is possible to design the ZFN chain so that it recognizes a specific section of DNA. This makes it possible to target specific areas within complex genomes.

Zinc fingers domains are found inhuman transcription factors - proteins that regulate the synthesis of RNA with a DNA template. When creating artificial nucleases, you can construct a chain of "zinc fingers" so that it will recognize a specific section of DNA.

If such a chain is long enough, itcan recognize relatively extended DNA sequences consisting of a number of trinucleotide fragments. This means a real possibility of targeted impact on specific areas within large complex genomes.

However, the method of "zinc fingers" was found to beserious disadvantages: firstly, it is not quite strict recognition of trinucleotide repeats, which leads to a noticeable number of DNA cleavages in “off-target” regions.

Secondly, the method turned out to be very laborious andexpensive, since for each DNA sequence it is necessary to create its own optimized protein structure of zinc-finger nucleases. Therefore, the system "zinc fingers" is not widespread.


In 2011, the journal Nature Methods named the systemTALEN (Transcription Activator-like Effector Nucleases) "method of the year" due to a wide range of possible applications in different areas of fundamental and applied science.

TALEN is one of the methods of targeted applicationbreak in DNA with its subsequent "healing" - to turn off genes in mice. Immediately after them, this technology was used to introduce a mutation into the mouse genome, leading to the development of one of the hereditary syndromes. The authors of the method of modeling genetically determined diseases managed not only to "spoil" the mouse genome, but also to correct it back.

  • CRISPR / Cas9

The method provides an accurate effect on specified DNA regions and can be used in almost any modern molecular biological laboratory.

This system is based on special areasbacterial DNA - CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats, or short palindromic cluster repeats). These repeats are separated by spacers - short fragments of foreign DNA. The latter are incorporated into the genome after DNA recombines with its genome.

Human Editing Examples

  • Genome editing right in the body

Disease of 44-year-old Arizona resident BrianMado manifested itself in early childhood. It is incurable and is inherited mainly by men. Mucopolysaccharidosis type II is a metabolic disorder: people with it have a mutation in a gene responsible for the production of an enzyme that is involved in the breakdown of complex carbohydrates. As a result, they accumulate in cells and cause numerous organ pathologies.

The man decided to take part in the clinicaltesting a new method - gene therapy. This is only the first phase of the study, and there should be three of them before registration of therapy (that is, before permission to use this method for all patients with Hunter syndrome).

The method used in Brian's caseMado, allows you to edit the genome right in the human body - and at the same time accurately enough to get into a specific piece of DNA. Editing takes place using the so-called "zinc fingers".

  • Genetically modified children

Chinese researcher He Jiankui edited the genomes of human embryos before in vitro fertilization, resulting in two children with altered DNA.

CRISPR / Cas9 system researcheredited the genomes of seven couples embryos during reproductive treatment. As a result of one of the pregnancies, two twin girls with altered DNA were born from a healthy mother and an HIV-infected father. He Jiankui explained that he removed the CCR5 gene from children, which gave them lifelong immunity to HIV.

  • Returning vision with gene therapy

To restore vision, optogenetic technologies can be used, with the help of which the work of neurons can be controlled with the help of light-sensitive proteins of bacteria and laser flashes.

Guided by this idea, biologists have created a virus,which can penetrate the ganglionic neurons. These nerve cells are responsible for transmitting signals from the retina to the human brain. Once in the ganglionic neurosis, a virus causes it to produce similar signaling molecules. However, this procedure does not restore vision by itself, since the proteins of bacteria react to light differently from the rods and cones of the retina.

To solve this problem, the Basel professorBotond Rosca University and University of Pittsburgh professor José Sahel have created special glasses that transform the incoming image into a format understandable to the brain and stimulate ganglion cells with laser flashes. As a result, the patient can see the silhouettes of large objects and objects and perform other complex actions.

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