Smithsonian Magazine Special Report
New gene editing technology enables scientists to eliminate carriers of malaria and Zika virus. But should they use it?
Jerry Adler; Photo by David Yoder
To the naked eye, the egg of Anopheles gambiae is just a black spot, but under a 100X microscope, it is a plump, slightly curved cucumber with a narrow end. In the wild, it usually appears in shallow, sun-drenched puddles in sub-Saharan Africa, but it can survive in any humid place around 80 degrees Fahrenheit. In a laboratory in London, Andrew Hammond, a PhD student in molecular genetics, used a small paintbrush to pick up a ball of Anopheles eggs behind three sets of closed negative pressure protection antechamber doors, and put them Arranged on a microscope slide. Hammond looks for the narrow end where the germline cells that will form the next generation are located. With the subtle push of the joystick, he manipulates a thin needle in his field of vision until it just penetrates the egg membrane, and then presses a button to release a one-minute DNA jet. Whether the genetic material reaches and binds to its target area is a matter of luck, and luck is usually in the mosquito. Hammond's proud success rate is about 20%.
This article is from the June issue of Smithsonian Magazine
A. gambiae is known as the most dangerous animal in the world, although strictly speaking, it only applies to females of this species. Females suck blood and hurt them only indirectly. Its bite is a small trouble unless it happens to transmit the malaria parasite Plasmodium falciparum, because it is the main transmission vector for humans. Although a huge international effort has reduced malaria mortality by about half since 2000, the World Health Organization estimates that there were more than 400,000 deaths in 2015, mainly in Africa. Children are especially vulnerable. The Bill and Melinda Gates Foundation has prioritized malaria in its more than $500 million pledge to fight infectious diseases in developing countries. Part of this money eventually went to Andrea Crisanti's laboratory at Imperial College London, a short walk from Harrods.
Crisanti is a messy, melancholy man with a gentle smile, trained as a doctor in Rome. Later, after studying molecular biology in Heidelberg, he developed a lifelong interest in malaria. About 30 years ago, after he concluded that the best way to eradicate the disease was to attack mosquitoes rather than parasites, he started tracking Plasmodium gambiae. "The vector is the Achilles heel of the disease," he said in a soft Italian accent. "If you [with drugs] track pathogens, all you do is develop resistance."
For more than a century, humans have been at war with members of the mosquito family, because the pioneering epidemiologist Sir Ronald Rose proved the role of Anopheles in malaria, and US Army Major Walter Reed was against Aedes aegypti and yellow fever. Ill made a similar discovery. The war is fought with shovel and insecticide, mosquito repellent, mosquito nets and fish that eat mosquito larvae, mosquito nets, screen windows and rolled newspapers. But all these methods are self-limiting. Rain filled the puddles again; insects evolved resistance to pesticides; predators could only eat so much.
By the time Crisanti joined Imperial College in 1994, molecular genetics proposed a new method, which he soon adopted, and his laboratory is now one of the most advanced in the world. Scientists have discovered how to insert beneficial mutations—such as the Bt gene—a natural pesticide—in crops such as corn. So, why not create a deadly mutation and insert it into the mosquito's DNA? One problem is that mosquitoes do not breed in factories, because commercial corn is increasing. In the wild, mosquitoes mate randomly and reproduce through Mendelian inheritance, which indicates that mutations spread slowly, if any. Unless artificial mutations convey some powerful evolutionary advantages—and the focus is on doing the opposite—it is likely to disappear.
In 2003, Austin Burt, a colleague of Crisanti at Imperial College, proposed a solution: Combine the required mutations with a "gene drive" that covers the ordinary processes of inheritance and evolution. Recall that genes are spelled out by DNA sequences woven into chromosomes, and chromosomes appear in pairs (23 pairs for humans and 3 pairs for mosquitoes). "Gene drive" involves copying a mutated gene from one chromosome to another. The point is that when a pair splits to form an egg and sperm, it doesn't matter which chromosome is passed-the engineered gene will be there. Therefore, in theory, a single mutation would be "driven" into almost every mosquito in the breeding population. Over the next ten years, Crisanti collaborated with a senior researcher named Tony Nolan and others, obsessively pursuing variants of this method, designing a genetic mutation that would cause female infertility, and another genetic mutation. Will cause males to dominate. The challenge is to create specific gene drives that replicate these mutations—the tedious, multi-year process of building custom DNA cutting enzymes.
Then, in 2012, Jennifer Doudna, a researcher at the University of California, Berkeley, and her colleagues developed a revolutionary new technique for editing DNA. For many years, researchers have known that certain genes in bacteria have short, repetitive chunks of DNA. (CRISPR stands for "clustered regularly spaced short palindromic repeats.") When a virus invades, the bacteria will copy part of the virus's genetic code and insert it into the space between the repeated CRISPR blocks. The next time the bacterium sees that code, an enzyme called Cas9 will guide its RNA to the exact sequence in the invading virus's gene. It cuts the DNA with incredible precision and re-fuses the strands together. Doudna and her colleagues used this process in the lab to quickly and easily edit any part of the gene they targeted. The following year, different teams led by MIT bioengineer Zhang Feng and Harvard University George Church showed that it can work in living cells.
The difference between CRISPR-Cas9 and other gene editing technologies lies in its versatility and accuracy. Unlike the custom enzymes that Crisanti and his team have painstakingly built, Cas9 seems to work in any type of cell. Researchers see the significance of treating genetic diseases, improving agriculture, and more sinister applications (such as making biological warfare agents). CRISPR also brings Crisanti's dream closer to reality. Now, he and his team can program Cas9's guide RNA to pinpoint any part of the gene and transfer the material they want to replicate.
If Crisanti's method works, in theory you can wipe out the entire mosquito. You can eliminate every type of mosquito, although you need to eliminate one at a time, and there are about 3,500 mosquitoes, of which only about 100 can only transmit human diseases. You may wish to stop less than a dozen species in the three genera-Anopheles (translation: "useless", malaria mosquitoes), Aedes (translation: "unpleasant", yellow fever, dengue fever, and Zika) The main transmission vector of the virus) and Culex mosquito (translation: "mosquito", responsible for spreading West Nile, St. Louis encephalitis and other viruses).
For thousands of years, the expanding population of Homo sapiens has caused the extinction of other species by eating other species, shooting them, destroying their habitat, or accidentally introducing more successful competitors into their environment. But scientists have never deliberately done so with the support of public health. This possibility raises three difficult questions: Will it work? Is it ethical? Will it have unforeseen consequences?
Crisanti's London laboratory is studying the feasibility of where the injected eggs will hatch into larvae. Genes that carry mutations are identified by "marker" genes, which glow under a microscope when viewed under certain light. Then put the mutant of interest back into the warm and humid air of the mosquito house, and put it back into the stacking tray with a white plastic mesh wall. On one side, there is a long sock-like tube, usually tied into a knot, through which the researcher can insert an aspirator to gently aspirate the sample. If you hold your hand, the female will feel the blood approaching and will gather on that side. When their blood meal time comes, it will nourish about a hundred eggs laid by a female at a time. An anesthetized mouse is placed on the roof of the cage with its abdomen down, and the female flies up and bites through the mesh. (Males feed on nectar and fruit in the wild, and feed on an aqueous solution of glucose, sucked out of a small glass bottle.) These insects live one month longer in the controlled environment of the cage than in the wild, and they usually don’t survive one or two. Weeks.
The next phase of research will be carried out in Perugia, Italy, which is home to one of the oldest universities in the world (founded in 1308) and a small elite research alliance, Polo d'Innovazione Genomica. A few miles from the winding alleys of medieval hilltop villages, in a glass-walled building on a turbulent square is the Baltic safety laboratory, with six high-ceiling "field cages", each with an area of 50 or 60 Square feet. A sign on the door warns visitors who may have been exposed to malaria because if a mosquito bites them, they may infect an escaped mosquito. The air inside is tropical. The females are not live rats, but are fed on small plates of cattle blood, heated to body temperature and covered with paraffin, giving them something to land. Females are attracted by the pheromone in human sweat, especially the pheromone on the feet. The laboratory staff said that they sometimes wear socks all weekend and take them to work on Monday to rub them on the feeding pan.
Inside, the lights change to simulate 24-hour tropical weather, and environmental cues trigger swarming behavior that is essential for mating. "This is the number of insects mating," explains chief entomologist Clelia Oliva. "Males flock in groups, females fly around in flocks looking for mates, and then gather together in the air. If you can't replicate it, you can't be sure whether your strain will succeed in the wild." Oliva is now While speaking, a fugitive who had escaped from the cage passed by Oliva, and she drove it out with her perfect slap while studying mosquitoes on Reunion Island in the Indian Ocean.
Researchers are skeptical about whether it is possible to eliminate mosquitoes. "I think it's a bit far-fetched to wipe out entire species on a global scale," said Steven Giuliano, an ecologist at Illinois State University. However, he added, "I think they are likely to reduce the local population, and may even eliminate a local species."
Other creatures have done similar things. Beginning in the 1950s, American entomologists Edward F. Knipling and Raymond C. Bushland wiped out an agricultural pest, spiralworm, from most parts of the United States and Central America. Their method is called "Sterile Insect Technology" and involves breeding and hatching millions of flies, sterilizing males with low-level gamma rays, and releasing enough numbers to inundate wild populations. Females mated with sterile males produce sterile offspring. It took decades, but it worked—the two won the World Food Prize in 1992—and now the same technology is used to control the Mediterranean fruit fly outbreak.
However, when trying to use sterile insect technology for mosquitoes, the results were mixed. It requires the released males to successfully compete with their wild counterparts in mating, and there is evidence that in mosquitoes, the same radiation that makes them sterile may also impair their mating behavior. No matter what the female mosquitoes are looking for in their mates, these male mosquitoes seem to have none.
Therefore, researchers have also been studying variants of sterile insect technology that do not require radiation. The British biotech company Oxitec has launched a pilot project in Piracicaba in southeastern Brazil. The target insect is the aegypti mosquito, which is the chief culprit in the transmission of yellow fever, dengue fever and other viral diseases. In the past six months, this work has become more urgent, because the aegypti mosquito is also a carrier of the Zika virus, blame Because of the terrible birth defects that broke out in the Americas.
In Oxitec's plan, male larvae with lethal mutations are raised in water with the antibiotic tetracycline, which can inactivate lethal genes. When these males mate with wild mosquitoes, their offspring die before they reproduce due to lack of tetracycline. CEO Hadyn Parry claimed in five studies covering relatively small areas in Brazil, Panama and the Cayman Islands that "the suppression rate of wild populations exceeds 90%." Now, the company hopes to expand into the subtropical regions of the United States, and recently passed a key regulatory hurdle to bring the plan to the Florida Keys.
Oxitec's technology predates CRISPR, and it does not use gene drives. Its goal is not to eliminate the Aedes mosquito, but to reduce the local population to a place where it can no longer be a vector of human disease. Of course, this is a temporary solution to a long-standing problem. Mosquitoes usually do not leave their hatching place more than a few hundred yards, but people do, and they can carry yellow fever. Mosquitoes can travel the world by plane and ship. Aedes albopictus, the "Asian tiger mosquito", arrived in the Western Hemisphere a few years ago, probably through tire transportation, spreading many of the same diseases as the Aedes mosquito. Therefore, even if the Oxitec plan is successful, it may need to be repeated every once in a while. "You begin to understand why Oxitec is a business," an American entomologist said dryly.
How the revolutionary CRISPR-Cas9 technology allows scientists to insert sterility genes into mosquitoes-so the genes are "driven" into the population, which ultimately leads to its extinction:
Scientists created the genetic code that disrupts the reproduction of female mosquitoes and injected customized DNA into fertilized mosquito eggs.
As insects develop, engineered genes are integrated into cells that produce male sperm and female eggs.
Mosquitoes have a total of three pairs of chromosomes (humans have 23 pairs), but a sperm or egg cell contains only one member of each pair of chromosomes. In mutated insects, the engineered gene (orange) is now part of the sperm or egg chromosome.
When mutated mosquitoes mate with wild insects, the chromosomes of their offspring will pair. The modified DNA contains highly targeted editing enzymes, which helps to insert changes into wild chromosomes. From left to right:
A mosquito inherited a chromosome from each parent.
The Cas9 enzyme cuts a gene on the wild chromosome.
Wild chromosomes use the changed genes as templates for self-repair.
Now both chromosomes in this pair of chromosomes carry mutations.
As the genes on the two chromosomes change, it will become more common in the population (as opposed to natural mutations that lack gene drive mechanisms). The altered gene (shown as a circle, on the right) is carried by male mosquitoes (orange), which maintain fertility. Females who inherit this change from both parents are sterile.
There is no doubt that eradicating Anopheles gambiae and Aedes aegypti will save many lives, and for most people, this is a good reason. "If the local populations of these species are eliminated, I don't think the world will get worse," said Giuliano, "and apart from eliminating the variola virus, it won't bother me." Even the most famous entomology in the world Home, the great environmentalist EO Wilson also said that he would not mourn for A. gambiae. "Keep their DNA for future research," he said, "and then let them go."
Nevertheless, there are voices demanding slow progress. Henry Greely, a professor of law and bioethicist at Stanford University, said: "If we intentionally cause a species to go extinct, we should think about it." "Before we take this step, I hope there are some considerations. And reflection, and social consensus.” His argument is partly based on a landslide: If it is a mosquito, why not a mouse? "I'm not sure if I care if mosquitoes will suffer or if they will suffer. But mammals or birds, I do care."
But suppose the target is the malaria parasite itself. As a single-celled protozoan, its sympathy for us is even smaller than that of insects? At the University of California, Irvine, geneticist Anthony James has been working on breeding mosquitoes since the 1980s. Although these mosquitoes can survive on their own, they do not transmit Plasmodium falciparum. The virus has a complex life cycle, from the mosquito's intestines to the circulatory system to the salivary glands, it takes up to three weeks to spread the virus. James realized that if he could give mosquitoes a gene that produces antibodies to Plasmodium falciparum, he could eliminate the parasite without killing an insect. He created the gene for antibodies, but he needed a way to spread it in the wild.
Then he heard about CRISPR-Cas9—especially the work of a molecular biologist named Ethan Bier at the University of California, San Diego, who recently introduced mutations into fruit flies. Bier acknowledged that in some cases, it may be necessary to remove genera like A. aegypti from large non-native parts of the world. However, whenever possible, he prefers less invasive methods. "I like this way of changing mosquitoes instead of making them go extinct," Bill said. "We have done enough. As a person, I don't want to participate in the eradication of a species, or even insects." James has successfully designed the antibody-producing genes and is studying gene drives. He can prepare insects for field testing within a few months, but he cannot predict how long the approval process will take. "We will not do anything stupid," he said.
If society chooses to eliminate one or more mosquitoes, what are the disadvantages? Mosquitoes play a key role in a few environments, such as the Arctic tundra. They hatch out of billions in a short period of time and are an important food resource for birds. Biologists believe that in most other places, ecosystems can survive the loss.
Nevertheless, according to Nolan, "Our goal is not to eliminate malaria mosquitoes from the earth. If we succeed, people won't even notice. There will be a lot of mosquitoes."
It is possible, even possible, that another species will replace the mosquitoes we have eliminated. For example, A. aegypti can be replaced by mosquitoes from the Culex pipiens species complex. Culex mosquitoes are the vectors of West Nile virus. "When the Aedes mosquitoes are present, it behaves very badly," Giuliano pointed out, but it may thrive without the Aedes mosquitoes. On the other hand, the newcomer may be a relatively harmless species; the niche of mosquitoes does not require them to carry diseases that are fatal to humans. In the long run, pathogens may evolve to be transmitted by mosquitoes that still exist, but humans have enough time to worry about this.
It can be said that the greater concern lies in the use of CRISPR itself and the powerful force it releases to the environment. "We can transform the biosphere into what we want, from mammoths to mosquitoes that don't bite," Greeley mused. "What should we think of this? Do we want to live in nature or in Disneyland?" Another concern is that CRISPR has put a potential weapon in the hands of terrorists, which can be used by terrorists to design epidemics. “Just like gene drives can make mosquitoes unsuitable for transmitting malaria parasites, they can be designed to carry cargo gene drives that can be used to provide deadly bacterial toxins to humans,” warns David Gurwitz of Tel Aviv University .
The National Academy of Sciences, Engineering, and Medical Sciences fully considered the threat, so it held a meeting last fall to discuss the impact of gene drive technology on biosafety. But many scientists think this is an excessive worry (and another horror movie scene where a high school student uses CRISPR to make a dog that glows in the dark in the basement). MIT ecologist Kevin Esvelt (Kevin Esvelt) said: "The gene drive of mosquitoes can be a very bad biological weapon." "They are slow [compared to spreading deadly microorganisms], they are easy It was discovered, and it was easy to establish a reversal mechanism."
But Esvelt has other ethical issues about using CRISPR technology in animals: "We will design their ecosystems without people in other parts of the world knowing or agreeing. We assume by default that what we design will not spread. Then assume they will spread. Normally you can make any fruit flies you want-natural selection will wipe the floor with them. But once you think about gene drive technology, you have to assume that whatever you make will happen once you leave the lab Spread. If it is not intentional human behavior, human error will prevail."
However, Esvelt himself is already considering whether and how to use CRISPR gene drives in mice (the main animal reservoir of Lyme disease) and mammals one day. He will design the local population to carry antibodies against the bacteria that cause Lyme disease. (This disease is transmitted from rats to humans through tick bites.)
If CRISPR works in mice, it will almost certainly work in humans. The least controversial application is for hereditary diseases, such as muscular dystrophy-this is likely to involve the repair of somatic (non-reproductive) cells in children or adults. But Chinese scientists have just announced the results of their second CRISPR study in human embryos. (They used non-viable embryos from a fertility clinic.) The results showed that there were "serious obstacles" to the method, but the technology is rapidly improving. For example, Harvard scientists recently modified the CRISPR method so that it can change a single letter of the genetic code, making it easier to prevent diseases such as Alzheimer's and breast cancer. CRISPR also opened the Pandora's Box for editing germline cells that pass genetic material to offspring. This may be of great benefit to the few people who carry genes for diseases such as Huntington's disease. More problematic is that it may encourage parents to customize their offspring, delete unnecessary but not life-threatening genes (such as lactose intolerance), or add genes with characteristics such as athletic ability, longevity, or intelligence.
This possibility has caused a lot of anxiety in the "playing God" column, and of course it should be taken seriously. Leaving aside philosophical objections, the actual disadvantage is that we don’t know all the genes that actually make people smarter (or taller, stronger, healthier, faster, etc.), and the only way to determine the answer It is trying different combinations on various embryos and waiting for them to grow up. At that time, if we get it wrong, it will be too late to fix, especially for unknowing subjects.
In the eyes of most ethicists, this is an unsolvable problem. Many of these issues were broadcast at the International Human Gene Editing Summit in Washington in December last year, revealing the differences between the medical community. The medical community hopes to help patients here and now, while some researchers worry about the influence of tabloids on the headlines. The news announced the birth of the first Franken baby.
At the same time, mosquitoes flew around in villages and cities in Central Africa, silently landed on sleeping children and bit them. The fight against malaria has made great progress in the past decade, but the price paid may not last indefinitely. In the Western Hemisphere, the threat of Zika virus has led to extraordinary measures, including warnings to women throughout South and Central America to consider delaying childbirth. This summer will tell us whether this disease will occur in the United States where two species of Aedes inhabit—Florida and the Gulf Coast, which may expand as the winter warms due to climate change. (The second of these two species of Aedes americana, Aedes albopictus, is a confirmed carrier of the virus and can be found in northern New England.) Public health officials are ready to welcome the possibility of a large number of infants contracting the virus . A devastating diagnosis of microcephaly and related brain injury. It is human transportation technology that spread these diseases on a global scale. Now, technology is providing a way to contain them, or even defeat them completely, and at the risk of unleashing powerful forces, we can only vaguely predict their impact.
Will we do this-we humans, a species with a ruthless desire for knowledge? The fruit of that particular tree has never been left uneaten for a long time. As far as Crisanti is concerned, he is ready to take over. "I want to see malaria eliminated in my lifetime," he said softly. He is 61 years old.
Mosquitoes: the story of the deadliest enemy of mankind
Jerry Adler is a former Newsweek editor.
Photographer David Yoder is based in Milan and Rome. His works often appear in "National Geographic" magazine and have been published by "New York Times", "Wall Street Journal", "Forbes" and "Newsweek".
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