基因编辑 z-bo 2020-08-13 476

　　When viruses attack bacteria, the bacteria will make a defensive response targeting DNA. Biologists use this mechanism to develop genetic engineering techniques.

　　If only a report is published, it will only get some attention. But when six reports are published at the same time, it means that it is the general trend.

　　Bacteria can also get sick, which is a potentially big problem for the dairy industry. The dairy industry generally relies on bacteria (such as Streptococcus thermophilus) to produce yogurt and cheese. Streptococcus thermophilus breaks down lactose in milk into pungent lactic acid. However, certain viruses, such as bacteriophages, can gradually weaken the bacteria, and then cause serious damage to the quality or quantity of food produced under the action of the bacteria.

　　CRISPR technology

　　In 2007, scientists from Danisco (a food additives company headquartered in Copenhagen, Denmark, currently acquired by DuPont) found a way to enhance the ability of bacteria to defend against phages. This discovery allowed DuPont to cultivate stronger strains for food production. Some basic principles have also been revealed: bacteria have a highly adaptive immune system that allows them to repel multiple attacks from a certain phage.

　　Suddenly, not only food scientists and microbiologists, but many fields have realized the importance of the bacterial immune system because it has a very valuable feature: it targets a specific gene sequence. In January this year, four research teams reported on this system called CRISPR. In the next 8 months, many scientific research teams used it to delete, add, activate or inhibit target genes in human, mouse, zebrafish, bacteria, fruit flies, yeast, nematodes, and crop cells, thus demonstrating this technology. The wide applicability.

　　George Church of Harvard University in the United States said that biologists have recently developed some new methods to precisely manipulate genes, "but CRISPR's efficacy and ease of use are superior in every respect." Church's laboratory was one of the first laboratories to apply this technology to human cells.

　　Based on CRISPR, scientists can build mouse models of human diseases much faster than before, and study single genes faster, and can easily change multiple genes in cells at once to study their interactions. But the craze for research on CRISPR may fade this year, as the limitations of this approach are beginning to show. But Church and other pioneers of CRISPR have established companies hoping to use this technology to treat genetic diseases. Blake Wiedenheft, a biochemist at Montana State University in Bozeman, USA, said: "I don't think there are any examples in any field that can show that this technology is developing too fast."

　　Stagger

　　This new genetic engineering tool was first reported in 1987 when a research team observed a strange repetitive sequence at one end of a bacterial gene. This phenomenon did not attract too many people's attention at the time. Ten years later, biologists deciphering microbial genomes often find similar puzzling patterns (a DNA sequence followed by a sequence that is almost identical but constructed in the opposite direction). This pattern occurs in more than 40% of bacteria and 90% of archaea.

　　Many researchers assumed that these strange sequences are meaningless, but in 2005, three bioinformatics teams reported that the spacer DNA usually matches the gene sequence of the phage, indicating that CRISPR may play a role in microbial immunity. “This is a very important clue,” said Jennifer Doudna, a biochemist at the University of California (UC) Berkeley. Eugene Koonin of the National Center for Biotechnology Information in Bethesda, Maryland, and his colleagues suggested that bacteria and Archaea take up phage DNA and store it as a template for RNA molecules (which prevent foreign DNA from matching), just as eukaryotic cells use a system called ribonucleic acid interference (RNAi) to destroy RNA.

　　In 2007, Rodolphe Barrangou, Philippe Horvath and others of the Danisco team demonstrated that they can change the resistance of Streptococcus thermophilus to phage attack by adding or deleting spacer DNA that matches the phage DNA. At that time, Barrangou (currently at North Carolina State University in Raleigh, USA) did not realize the full potential of CRISPR. He said: "We still don't know whether these elements can become readily available technologies like the compelling gene editing technology."

　　Doudna took the next step with Emmanuelle Charpentier, who currently works at the Helmholtz Infection Research Center and Hanover Medical School in Germany. They independently combed the roles played by various CRISPR-related proteins, and studied how the spacer DNA plays a role in the immune defense of bacteria. But the two experts quickly turned to a CRISPR system that relies on a protein called Cas9, because this CRISPR system is simpler than other CRISPR systems.

　　When encountering a phage invasion, CRISPR will respond. At this time, the bacteria transcribes the spacer DNA and DNA palindrome into a string of long RNA molecules. tracrRNA (an additional RNA fragment) and Cas9 work together to produce crRNA (RNAs derived from the spacer). Charpentier's team reported this discovery in the journal Nature in 2011. The team proposed that Cas9, tracrRNA and crRNA would attack the foreign DNA paired with crRNA in some way.

　　ravage the world

　　Speed is not the only advantage of CRISPR. Church's team is promoting the use of TALENs (synthetic nucleases) in human cells. Among the three types of human cells, the CRISPR system is more efficient than TALENs in cutting target DNA and can process more genes than TALENs. To illustrate the simplicity of the CRISPR system, Church's team synthesized thousands of guide RNA sequences-which can lock 90% of human genes.

　　An independent research paper (completed by Feng Zhang, a synthetic biologist at the Broad Institute in Massachusetts, USA, and his colleagues) that appeared almost at the same time as Church’s paper showed that CRISPR can immediately lock and cut two genes in human cells. In collaboration with Rudolf Jaenisch, a developmental biologist at the Whitehead Institute of Biomedical Research in Massachusetts, Zhang split five genes in mouse embryonic stem cells.

　　These work laid the foundation for breeding mutant mice, which is a key tool for biomedical research. One method is to implant embryonic stem cells from mutant mice into a growing embryo. His team found that this could simply inject Cas9 messenger RNA and two guide RNAs into a mouse's egg or fertilized egg.

　　According to Zhang's CRISPR technology, a new mouse model is about to be tested within a few weeks. Zhang believes that this method is not limited to rats. As long as you can manipulate the embryo and reimplant it, you will be able to carry out the research on larger animals (even primates).

　　Three weeks after Zhang and Church’s report was published online, Doudna’s team and a South Korean research team reported that they successfully used CRISPR to excise the DNA of human cells. At the same time, another team revealed that they used CRISPR to create mutant zebrafish. A series of research reports have created a synergistic effect, which has won widespread attention for the biological community. Charles Gersbach, a biomedical engineer at Duke University in Durham, North Carolina, said: "If only one report is published, it will only get some attention. But when six reports are published at the same time, it means that it is the general trend. "

　　A year ago, when Gao Caixia saw Doudna and Charpentier's research report, she was convinced by their theory. Gao Caixia's team comes from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing. They have used zinc finger structure and TALENs technology to conduct research on rice and wheat. By using CRISPR, her team has successfully disabled four genes in rice, which means that the technology can be used to improve rice, an important crop. As for wheat, they deleted a gene that gave the wheat powdery mildew resistance. The progress of CRISPR is exciting. The research report of Gao Caixia's team was published in the August issue of "Nature-Biotechnology". At the same time, four other reports on the research results of CRISPR in plants and mice were published at the same time.

　　The cost of using CRISPR is very low: free software makes the cost of designing guide RNA (for specific genes) zero, and it only costs $65 to obtain genes from the gene resource library called Addgene to design your own CRISPR system. Since the beginning of this year, Addgene (a total of 11 scientific groups have provided it with DNA sequences that can be used in the CRISPR system) has witnessed the creation of 5000 CRISPR constructs. In July of this year, Addgene received 100 orders (in order to design a new structure) within a week. Addgene's executive director Joanne Kamens said: "Addgene is on sale."