基因编辑 z-bo 2020-08-14 553

Three years ago, Bruce Conklin occasionally discovered a method that changed his research methods in the entire animal laboratory.

　　 Bruce Conklin is a geneticist at the Gladstone Institute in San Francisco, USA. He has been dedicated to studying how differences in DNA affect various human diseases, but his research tools are very laborious to use. When he processed the patient's cells, it was difficult to identify which sequence was the key to the disease and which sequence was just background noise. The introduction of mutations into cells is costly and arduous. "The workload of changing a gene is enough for a student to write an entire thesis," he said.

　　 Later, in 2012, he read about a newly published technology called CRISPR. CRISPR enables researchers to quickly change the DNA of any tissue-including human tissue. Immediately, Conklin abandoned the original method of building disease models and switched to this new method. Today, his laboratory is keen to change a variety of genes related to heart disease. "CRISPR has changed everything," he said. CRISPR is leading an upheaval in biomedical research-a view that has gained wide acceptance. Unlike other gene editing technologies, CRISPR is cheap, fast and easy to operate, so it quickly swept laboratories around the world. Researchers hope to use it to modify human genes and eliminate diseases, cultivate stronger plants, eliminate pathogens, and achieve many other goals. "Since I was engaged in scientific research, I have seen two major breakthroughs: CRISPR and PCR." said geneticist John Schimenti from Cornell University. Since the advent of PCR in 1985, this gene amplification technology has set off a revolution in genetic engineering, and just like PCR, "CRISPR has affected life sciences in so many fields," he said.

　　 However, despite the many contributions of CRISPR, some scientists still worry that the rapid progress in this field does not give us enough time to discuss the ethical issues and safety hazards that such experiments may cause. In April, the news that scientists used CRISPR to transform human embryos (Nature 520, 593–595; 2015) pushed this issue to the forefront. Although the embryos they used could not develop into babies, this report still sparked fierce controversy, including whether CRISPR should be used to genetically modify the human genome, or how such modifications should be made. Moreover, there are other concerns. Some scientists hope to have more relevant research to explore whether the technology will cause unexpected and potentially harmful genome changes, while others worry that changing organisms may disrupt the entire ecosystem.

　　 "In the laboratory, it is quite easy to acquire this capability-you don't need very expensive equipment, and the personnel don't need to be trained for many years," said Stanley Qi, a systems biologist at Stanford University in California. "We should think carefully about how to apply this ability."

　　Research revolution in biological research

　　Biologists have long been able to use molecular tools to edit the genome. About ten years ago, they were excited about zinc finger nuclease, which is expected to edit the genome accurately and effectively. However, according to James Haber, a molecular biologist at Brandeis University in the United States, it costs more than US$5,000 to order zinc finger nucleases. Due to the cost and difficulty of operation, zinc finger nucleases have not been widely used. The working principle of CRISPR is different: it relies on an enzyme called Cas9. Cas9 uses a piece of guide RNA molecule to accurately locate the target DNA, and then it edits the DNA, deletes genes or inserts desired sequences. Researchers often only need to order RNA fragments, and other components can be purchased on the market. The total cost is only $30. "This has effectively contributed to the popularization of this technology so that everyone can use it," Haber said. "This is a huge revolution."

Soon, CRISPR technology eclipsed zinc finger nucleases and other editing tools (see "The Rise of CRISPR". For some people, this means abandoning the technology that they have spent years proficient in. "I'm very depressed. "Bill Skarnes, a geneticist at the Sanger Institute of the Wellcome Trust in Sinxton, UK, said, "But at the same time I am also very excited." Bill Skarnes put a lot of effort into the technology introduced in the mid-1980s: Inserting DNA into embryonic stem cells, and then using those cells to create transgenic mice. This technology has become a major tool in the laboratory, but at the same time it is time-consuming and labor-intensive. Compared with traditional technology, the time required for CRISPR is greatly shortened. Skarnes accepted it two years ago CRISPR technology.

　　In traditional biological research, researchers rely heavily on model organisms such as mice or fruit flies, partly because only these species have been equipped with a complete set of genetic modification tools. And now CRISPR makes it possible to edit the genes of more species. For example, in April this year, scholars at the Whitehead Institute for Biomedical Research in the United States released a study on Candida albicans using CRISPR. This fungus is particularly deadly to people with weakened immune systems. However, it has been very difficult in the laboratory. It is difficult to manipulate its genes. Jennifer Doudna, a CRISPR pioneer at the University of California, Berkeley, has collected a list of CRISPR-modified species. So far, she has registered more than 30 organisms, including a disease-causing parasite called trypanosome and yeast that can synthesize biofuels.

　　However, the rapid progress is also accompanied by defects. "People don't have time to identify some very basic parameters in this system," said Bo Huang, a biophysicist at the University of California, San Francisco. "There is a psychology: as long as it works, we don't need to understand its mechanism of action." This means that researchers occasionally encounter some trouble. In order to apply CRISPR to imaging research, Huang struggled with his laboratory members for two months. He guessed that if they had a better understanding of how to optimize the design of "guide RNA"-a basic but important detail-it might not have been so long.

　　 In short, researchers see this flaw as a small price to pay for a powerful technology. However, Doudna has deeper concerns about its safety. Her worries began at a conference in 2014, at which she saw a postdoc report on the work of modifying a virus to carry CRISPR components into mice. Mice ingest the virus through their breath, allowing the CRISPR system to make mutations and build human lung cancer models. Doudna shuddered; even if there is a slight difference in guide RNA design, the same thing will happen in the human lungs. "It's scary to think that you might have a student doing research like that," she said. "The point is that people have to understand what this technology can do."

Andrea Ventura, a cancer expert at the Memorial Sloan Kettering Cancer Center in New York and the first author of this work, said that his laboratory carefully considered the safety implications: they designed the guide sequence to be specific Sex targets the genetic region of mice, and the virus also has defects and cannot replicate. He agrees that even if the risk is minimal, it is important to prevent problems before they happen. "The guide sequence is not used to cut human DNA, but who knows," he said. "Probably there is no problem, but you still need to beware of this."

　　 eliminate disease

　　 Last year, Daniel Anderson, a bioengineer at MIT in Cambridge, and his colleagues used CRISPR in mice to correct mutations associated with the human metabolic disease tyrosinaemia. This is the first time that CRISPR has been used to correct disease-causing mutations in adult animals—an important step on the road to clinical gene therapy using this technology (see "A Brief History of CRISPR").

　　 The idea that CRISPR can accelerate the development of gene therapy has become a stimulant in the scientific community and the field of biotechnology. However, while Anderson's research shows the potential of CRISPR technology, it also points out that there is still a long way to go to achieve clinical applications. In order to transfer the Cas9 enzyme to the target organ-the liver, the team must pump a large volume of fluid into the blood vessels-a measure that is usually not feasible in the human body. This experiment only corrected pathogenic mutations in 0.4% of cells. For most diseases, this ratio is too low to have any effect.

　　 In the past two years, many companies can't wait to invest in CRISPR-based gene therapy. Anderson and others said that the first batch of such clinical treatments may come out in the next one or two years. The first treatments are likely to occur in situations where CRISPR components can be injected directly into tissues, such as those in the eyeball; in another case, cells can be removed from the body, modified in the laboratory and implanted back into the body. For example, hematopoietic stem cells may be repaired to treat diseases such as sickle cell (anemia) or β-thalassaemia. The introduction of enzymes and guide RNA into a variety of other tissues will be a big challenge, but researchers hope that one day the technology can be widely used in various genetic diseases.

　　 However, many scientists warn that there is still a long way to go before CRISPR can be used safely and effectively. Scientists hope to increase the efficiency of editing genes, but at the same time ensure that they will not cause health-affecting changes in other parts of the genome. "These enzymes may cut to areas other than the restriction site you designed, which will have multiple effects," Haber said. "If you want to replace the sickle cell gene in someone’s stem cell, someone will ask you. ,'Well, what kind of damage might this cause in other parts of the genome?'"

　　Keith Joung, who studies gene editing at the Massachusetts General Hospital in Boston, has been developing technology that can track Cas9 off-target cuts. He said that for different cells and different sequences, the probability of such cleavage is very different: he and other laboratories have observed that the probability of such off-target is between 0.1% and 60%. He said that even if the probability of occurrence is low, if off-target cutting promotes cell growth and causes cancer, it may also be potentially dangerous.

　　Editas president Katrine Bosley said that since CRISPR has so many problems, it is important to control the expectations of CRISPR reasonably. Editas is a company located in Cambridge, Massachusetts, that develops CRISPR-mediated gene therapy. Bosley is an expert in applying new technologies to commercialization. She said that usually the difficulty lies in convincing others that a certain method is feasible. "But CRISPR is often the opposite," she said. "There are so many surprises and support, but we must also truly realize the efforts we need to make to achieve our goals."

　　 CRISPR in agriculture

　　 When Anderson and others worked on modifying genes in human cells, others targeted crops and livestock. Before the advent of gene editing technology, it was usually only possible to insert genes at random locations in the genome, as well as sequences from bacteria, viruses, or other species that promote gene expression. However, this process is inefficient, and those who are opposed to mixing genes from different species, or who are worried that inserted genes may interfere with other genes, often attack this technique. What's more, obtaining approval for genetically modified crops is troublesome and expensive. This has resulted in most genetically modified crops being large-scale commercial crops, such as corn or soybeans.

　　 CRISPR technology may change this situation: the convenience and cheapness of CRISPR make genome editing of special crops and livestock grown on a small scale feasible. In the past few years, researchers have used this method to transform petite pigs and breed disease-resistant wheat and rice. They have also made progress in transforming dehorned cattle, disease-resistant goats and high-vitamin sweet oranges. Doudna predicts that her list of CRISPR-modified species will continue to grow. She said, "This is a very interesting opportunity. You can consider experiments or genetic modification of plants that are not commercially important, but from a research perspective or as home gardening vegetables."

　　 The ability of CRISPR to precisely edit DNA sequences is conducive to more precise genetic modification. However, once implemented, it will also make it more difficult for managers and farmers to identify genetically modified species. "With gene editing technology, it is no longer possible to truly track genetically modified species," said Jennifer Kuzma, a science policy researcher at North Carolina State University in Raleigh. "To detect that a gene has been mutated through traditional methods, It’s genetically engineered and it will be very difficult."

　　 This is a wake-up call for genetically modified crops, and it also raises a problem for countries that are committed to controlling genetically modified plants and animals. In the United States, the Food and Drug Administration (FDA) has neither approved any genetically modified animals for human consumption, nor has it announced measures to regulate genetically modified animals.

　　 According to existing regulations, not all crops that have undergone genome editing need to be managed by the USDA (Nature 500, 389–390; 2013). However, in May, the Ministry of Agriculture began to seek ways to improve the management of genetically modified crops—a move that many people saw as a sign of the agency's reassessment of its role based on technologies such as CRISPR. "The window has been broken," Kuzma said. "I don't know what will fly in from the window. But breaking the window is very exciting."

　　 Transform the ecosystem

　　In addition to agriculture, researchers are still thinking about how CISPR can be applied to wildlife. One method that has been receiving much attention is called "gene drive", which can quickly spread the modified gene throughout the population. This work is still in its early stages, but similar technologies can be used to wipe out disease-causing mosquitoes or lice, eliminate invasive plants, or wipe out herbicide-resistant weeds that cause headaches for some American farmers.

However, many researchers have expressed deep concern about changing or eliminating the entire population, which may have profound and unknown effects on the ecosystem: for example, it may mean that other pests will grow, or it may affect predators at the top of the food chain. . The researchers also noticed that over time, the guide RNA may mutate, causing it to target other regions of the genome. This mutation will then spread throughout the population, with unforeseen consequences.

　　 "It must be highly rewarding because the risk is irreversible-and for other species, the consequences are unpredictable and difficult to calculate," said George Church, a bioengineer at Harvard University in Boston. In April 2014, Church collaborated with a group of scientists and policy experts to comment on Science, warning researchers of the risks involved, and making recommendations to avoid accidental spread of experimental gene drives.

　　At the time, gene drives seemed out of reach. However, within a year, Valentino Gantz, a developmental biologist at the University of California, San Diego, reported on such a system they designed in fruit flies. Bier and Gantz packed the fruit flies into a three-layer box and raised the level of laboratory safety to the level of dealing with malaria-causing mosquitoes. However, they did not follow all the guidelines put forward by the author in the comments, such as devising a plan to reverse genetic modification. Bier said that they were conducting the first theoretical verification experiment at the time, just wanting to verify the success of the system, and did not want to complicate things before that.

　　 For Church and others, this is an obvious warning that the routine use of CRISPR genome editing may have unforeseen and counterproductive results. "It is necessary for national regulators and international organizations to take charge of this matter-real responsibility," said Kenneth Oye, a political scientist at the Massachusetts Institute of Technology and the first author of the Science commentary. "We have to take more action." The National Research Council of the United States has organized a panel to discuss gene drives, and other high-level discussions are also underway. However, Oye is concerned that science and technology are developing rapidly, and that regulatory measures may not be introduced until after the gene drive has become widespread.

　　 This is not a question of right and wrong. Micky Eubanks, an insect ecologist from Texas A&M University in College Station, said that the idea of gene drive shocked him at first. "My first reaction was, ‘oh my god, this is terrible, it’s amazing.’" he said. "But when you think twice and weigh the environmental changes that we have caused and will continue to cause, gene drive is just a drop in the ocean."

　　 Some researchers have seen the lessons that CRISPR can learn from in other new technologies that were amazing and worrying, and then encountered trouble in the embryonic stage and finally disappointed. James Wilson is a medical geneticist at the University of Pennsylvania in Philadelphia. In the 1990s, he was at the center of thriving gene therapy-but it turned out that gene therapy declined after a clinical trial failed and killed a young man. . This field ended hurriedly and only recently began to recover. The CRISPR field is still young, Wilson said, and it may take several years to see its potential. "It's still developing. These ideas have yet to mature."

　　 However, Wilson once again became indissoluble with CRISPR. He said that unless his lab begins to play with CRISPR, he is skeptical of all relevant guarantees. "CRISPR will eventually show its skills in clinical treatment," he said. "It's amazing."