Gene editing 72 changes from homologous recombination to base editor

Genetic editing, especially the "Genetic Devil" CRISPR news report can be seen almost every day. In March alone, there were two heavy results that attracted widespread attention. First, David Liu, who worked with Zhang Feng to create the “genetic magic scissors” CRISPR technology, developed the cells using gene editing technology. The "Cell Recorder" (CAMERA) for taking pictures of the event. Just as the black box can record the state of the aircraft at the time of the accident, the police's portable camera can restore the scene. The “cell recorder” can record the changes in the activity of the cells after exposure to light, antibiotics and viruses.

Another major achievement comes from Chinese scientists. Researchers at the Shanghai University of Science and Technology and the Max Planck Institute for Computational Biology of the Chinese Academy of Sciences published a paper in Nature and Biotechnology published on the 19th, saying that they have developed a series of new base editors based on CRISPR/Cpf1 (Cas12a) (dCpf1). -BE), in theory, can correct the genetic mutations of hundreds of human diseases, so it has great potential for clinical application.

In recent years, CRISPR gene editing technology has even been selected into the Top Ten Scientific Breakthroughs of Science magazine twice. The term "genetic editing" has become a popular scientific term, but where does it originate, when it is popular, and why Amazing 18 martial arts? Most people are not "capable of" one or two.

Past gene knockout or knock-in

Since the American biologist James Watson and the British biologist Crick discovered the DNA double helix structure in 1953, people have been actively exploring efficient and convenient gene editing technology. So far, the development of gene editing technology has reached the third generation.

"Gene editing technology is a technology that can precisely modify the genome or transcript, and can perform site-directed mutagenesis, knockout or knock-in of fragments, etc." Academic Leader of Cardiovascular Research Center, Institute of Medicine and Health, Hebei Medical University (PI), Professor Sun Shaoguang, Department of Biochemistry and Molecular Biology, Basic Medical College, told the Science and Technology Daily reporter.

In the 1980s, gene targeting techniques based on the development of mouse embryonic stem cells and DNA homologous recombination technology revolutionized life sciences. With this technology, scientists can perform targeted knockout or knock-in on gene sequences such as mice. The first generation of gene editing technology was born.

Homologous recombination is a recombination between or within a DNA molecule containing non-sister chromatids or homologous sequences on the same chromosome. Among them, gene knockout is the deletion of the original in the genome; gene knock-in is the integration of genes that do not exist into the genome. Although scientists have won the Nobel Prize in Medical Physiology for this technology, it has certain limitations. "It has the disadvantages of low editing efficiency, difficult operation, long cycle, high cost, limited application range, etc." Sun Shaoguang said.

The second generation uses specific endonucleases for specific site cleavage

In order to overcome the limitations of the first generation of gene editing technology, such as low efficiency and difficult operation, in the 1990s, scientists developed a second generation gene editing technology relying on endonuclease, including zinc finger nuclease (ZFN) and transcription. Activation factor-like effector nuclease (TALEN) is the most mainstream representative.

ZFN is an artificially engineered restriction endonuclease that is a fusion of the zinc finger DNA binding domain and the restriction endonuclease DNA cleavage domain. They are able to recognize and bind to specific sites, efficiently and accurately cleave target DNA. Because of the natural DNA repair capabilities of cells that repair target gene breaks, researchers can edit specific genomic loci.

TALEN technology uses the DNA recognition module to target TALEN elements to specific DNA sites and bind them, then complete the cleavage of specific sites under the action of specific nucleases, and complete the genome editing by means of the inherent repair process in the cells.

This life magic scissors hand CRISPR and base editor

CRISPR is a mechanism for bacterial defense against viral invasion. In 2012, it was developed by scientists to become a popular tool for efficiently, accurately and programmatically modifying the genome of cells.

If we treat a viral infection as a time bomb, the bacteria only has a few minutes to dismantle the bomb before it "explodes." Therefore, many bacteria have an adaptive immune system called CRISPR that detects viral DNA and destroys it. The Cas9 protein can be targeted to cleave viral DNA under the guidance of a guide RNA. Based on this function of Cas9, scientists have developed it as a genetic manipulation technique to precisely delete or insert specific DNA fragments.

The reason why CRISPR/Cas9 technology is popular among many scientists is because it has a number of advantages. “Our laboratory is using the CRISPR/Cas9 tool to study the function of genes. It is easy to operate, low in cost, and has a short cycle. It can be operated in a routine laboratory and can be operated at the cellular level or at the level of animals and plants. It is widely used in medical, agronomy, microbiology and other research fields." Sun Shaoguang said.

In recent years, although the CRISPR/Cas9 gene editing technology has been widely used by scientists around the world, repairing point mutations has always been an unsolved problem. To this end, Liu Ruqian team developed a "base editor" that can exchange one base of DNA in the cell for another base to achieve the purpose of accurately editing the gene.

In middle school we know that the double helix of DNA consists of four bases: adenine (A), thymine (T), cytosine (C) and guanine (G). Among them, A and T pair, C and G pair, constitute the genetic information of human. The problem is that cytosine (C) is prone to deamination mutations, and as a result, CG becomes an AT combination. This single base variation results in hundreds of hereditary diseases, and the base editor can reduce the AT combination formed by the mutation into a CG combination, which has found a new direction for curing such genetic diseases.

The future will display 18 martial arts in research and application.

After the advent of gene editing technology, its application field has expanded rapidly, including the opening of new areas of basic research in molecular biology, the promotion of precise genetic modification of microorganisms, plants and animals, and gene therapy for major genetic diseases.

Gene editing technology can accurately and precisely modify the animal and plant genes, so that it not only does not affect the normal physiological functions of animals and plants, but also can get the animal and plant performance we want, such as high yield of crops, higher lean meat rate, etc. An excellent variety that humans need.

When studying the relationship between genes and diseases, scientists can also construct animal models of hereditary diseases through gene editing techniques. "This is of great significance for the study of hereditary diseases, which is why we have been paying close attention to gene editing technology since the first generation." Hu Yuhua, co-founder of Jiangsu Luling Gene Biomedical Co., Ltd., once said.

The most popular application areas for gene editing technology are gene therapy and drug discovery. Traditional gene therapy is to introduce normal gene fragments into cells to replace defective genes, but this method is difficult to locate accurately and may have great side effects. Gene editing technology can accurately correct or excise genes, which is undoubtedly the gospel of radical cure for genetic mutations. In the field of drug discovery, the team that uses CRISPR can bring customized therapies to market faster and accelerate the drug development process.