It is safer not to rely on the virus to sneak into the body "God's scalpel" April 24, 2019 Source: Science and Technology Daily Decoding gene editing When sending CRISPR-Cas9 into cells, it is necessary to use the help of a carrier. Among them, the viral vector is the most popular delivery method at present, but its cost is high, there is off-target effect, and it will cause cancer risk to the human body. The latest achievement of the Zhejiang University research team opened another window for the use of non-viral vectors in gene editing. Accurately locate and cut off genetic loci on DNA, shut down a gene or introduce new gene fragments, so that patients who have lost hope can regain the cure... CRISPR gene editing technology has been hailed as "God's surgery since its inception" Knife". However, when this magical "scalpel" actually has a miss, its off-target effect has always been one of the key obstacles to its application. Recently, researchers from Nanjing University, Xiamen University and Nanjing University of Technology have developed a new type of "genetic scissors" tool, which can achieve genetic editing and control, and has broad application prospects in the treatment of major diseases such as cancer. Currently, the results have been published in the new issue of the US Science Progress. Concerns from viral vectors Genetically modified soybean oil, insect-resistant cotton, and even genetically modified infants that are immune to AIDS... Although arguing, in just a few decades, genetic editing technology as a product of a new era is still rapidly established with most ordinary people. Contact. However, many people are not familiar with the tools and principles used. Since the secrets of the genetic code were revealed in the 1960s, human attempts to transform genes have never stopped. In a more vivid way, genetic editing can be understood as the use of "genetic scissors" to break the DNA strand, and the process of transforming the target DNA fragment, whether it is to add or knock out the gene, essentially changes the organism from the molecular level. Traits. At present, the most commonly used "genetic scissors" by scientists is a foreign DNA called CRISPR-Cas9, which is inseparable from bacteria. The virus uses its bacterial cell tools for its own gene reproduction services. In the process of fighting the virus, the bacteria evolved the CRISPR system in the body, and can remove the viral genes from their chromosomes without any sound. Scientists have used this feature to develop this cutting-edge "genetic scissors." How to send CRISPR-Cas9 to cells? This requires the help of the carrier. According to the source of the gene vector, we can classify the gene vectors into five major categories, namely plasmid vectors, phage vectors, viral vectors, non-viral vectors and microcircular DNA. Among them, viral vectors are currently the most popular delivery method. As of June 2018, more than 70% of the gene drug carriers in clinical trials were viruses. After the complex is attached to the virus, the virus invades the nucleus of the target cell, and the CRISPR-Cas9 "genetic scissors" can perform its true function. Retroviruses, adenoviruses, and adeno-associated viruses (AAVs), three major types of viruses, have been widely used in the treatment of genetic material. However, building viral vectors is a laborious and costly process, and delivery using these viral vectors is not foolproof. "The advantages of CRISPR-Cas9 are obvious, and the disadvantages are obvious. It has the off-target effect, and it may cut off other areas outside the target. When the normal area is cut, it will cause great damage." Modern Engineering of Nanjing University Said Song Yujun, a professor at the School of Applied Sciences. Studies have shown that viral vectors have inherent disadvantages in the CRISPR-Cas9 system, including cancer risk, insertion size limitations, and the production of immune responses in the human body. For example, retroviruses may cause insertional mutations that result in cancer, and high doses of AAV for gene therapy can also cause severe toxicity. A safer genetic editing vector is coming The safety of viruses as a transport vector for genetic engineering is not yet fully controlled, so scientists have proposed several alternative non-viral delivery materials, including gold nanoparticles, black phosphorus, metal organic frameworks, graphene oxide and various nanomaterials. . These materials have greatly improved in security compared to viruses. However, the time of gene editing and gene editing The process is still not controlled by scientists. Published in the latest scientific research results in the "Scientific Progress" magazine, researchers from Nanjing University, Xiamen University and Nanjing University of Technology developed a new non-viral vector of "genetic scissors" tool, which can be controlled by near-infrared light control. "Genetic methods can achieve controlled editing of genes in time and space in vivo, and have broad application prospects in the treatment of major diseases such as cancer. In response to the off-target effect of CRISPR-Cas9, the R&D team developed a non-viral vector called “upconversion nanoparticles†after a year and a half of experiments. The nanoparticles can be endocytosed by cells. The photoactive compound locks CRISPR-Cas9 on the upconverting nanoparticles. Song Yujun said: "Infrared light has strong tissue penetration, which makes it possible to apply genetic editing technology safely and accurately in deep tissue of the human body." The experimental triggering device consists of two kinds of light - near-infrared light and ultraviolet light. Near-infrared light and ultraviolet light have special properties, the former can penetrate the human tissue to reach the target position, and the latter can cut off the photosensitive molecules. Exposure to near-infrared light, these nanoparticles absorb low-energy near-infrared radiation and convert it into visible ultraviolet light, which automatically opens the "lock" between the nanoparticle and the Cas9 protein, allowing the Cas9 protein to enter the nucleus, thereby achieving the target The precise knockout of the point gene induces apoptosis of the tumor cells. The team verified the effectiveness of the system from the perspectives of genes, proteins and cells. During the treatment of tumor-bearing mice, the team found that only the tumors in the near-infrared light irradiation group were effectively inhibited. And from the size of the tumor removed after 20 days, the tumor in the experimental group was much smaller than the control group. This technology opens another door to the use of non-viral vectors in genetic engineering. Once the technology is available in the future, tumors, especially solid tumors, can achieve non-invasive treatment, and patients such as Parkinson's disease and diabetes can benefit from this technology. Non-viral vectors are infinite in the future Although viral vectors are widely used in clinical practice, their safety uncertainty, high preparation and transportation costs restrict their promotion in genetic engineering. Therefore, non-viral vectors are increasingly attracting the attention of researchers. "At present, non-viral carriers of various nanomaterials are being done by researchers, such as biodegradable biomaterials, and its prospects are very large." Song Yujun told reporters. There are two directions for the study of non-viral vectors, one is the organic material gene delivery system, and the other is the electrodeless material gene delivery system. In the field of organic materials research, liposomes, polyethyleneimine and its derivatives, cationic peptides, dendrimers and their derivatives, chitosan and its derivatives, polyurethanes, cyclodextrins and their derivatives The main direction of research for scientists. Non-viral lipid nanoparticles designed by various liposomes are easy to prepare, have less immune response, and have a larger payload. Therefore, they have been widely used in clinical practice, such as vaccine and gene drug delivery, cancer treatment, and tumor imaging. This kind of transmitter will be used in school. Delivery systems based on polyethyleneimine and its derivatives have also been used in clinical trials for multiple diseases, including ovarian cancer, pancreatic cancer, primary peritoneal malignancy, multiple myeloma, and the like. The research on other transmitter materials has not entered the clinical stage. Inorganic materials are easier to control than organic materials, and their size is adjustable and the surface is easy to modify. Gold nanoparticles, carbon nanotubes, graphene, upconversion nanoparticles and other materials have been extensively studied. The mainstream delivery methods include the formation of complexes between negatively charged genes and positively charged inorganic nanoparticles, and the connection of genes in responsive covalent bonds. The amphiphilic polymer is modified on the nanoparticle or on the surface of the inorganic nanoparticle, and the negatively charged gene is adsorbed in the polymer layer by electrostatic action. For the first time, the new technology of light manipulation gene editing developed by Song Yujun’s team. At present, the research of inorganic delivery materials is still in the laboratory stage, and the trials in the clinical stage have not yet been approved. There is no definitive conclusion about its impact on the organism. “Inorganic nanoparticles contain elements that are not essential to the human body, so some side effects may occur. In our experiments, the observation time on the mouse level was relatively short, as few as several weeks and several months, and there was no cell level or animal level. Discover the big impact. If you can find suitable, safe, inorganic materials with the same function, its prospects will be unlimited." Song Yujun is full of confidence in the future of non-viral carriers. Xi'an Healthway Biotech Co.,Ltd , https://www.xianhealthway.com