Edit the mushroom

The hundred farmers who packed the banquet hall of the Mendenhall Hotel in Chester County, Pennsylvania, may not have had a background in gene editing, but they understood mushrooms. These local growers produce an incredible amount of about half a million kilos of mushrooms every day, and this is one of the reasons that thanks to them Pennsylvania dominates the American market whose annual value is 1.2 billion dollars. However, some of the mushrooms they produce turn brown and rot on the shelves. If you've ever held a rotting, slippery, formerly white mushroom in your hand, you understand why no one will buy it. Mushrooms are so sensitive to physical damage that even careful picking and packing can activate an enzyme that accelerates their decay process.

On a foggy morning in the fall of 2015, during a training course on mushrooms, a biologist named Yinong Yang and report on a possible solution to the browning problem. Young, a cheerful and polite professor of plant pathology at the University of Pennsylvania, is not an expert in the field. (“The only thing I know about mushrooms is how to eat them,” he says.) But he edited the genome of Agaricus bisporus, the most popular edible mushroom in the Western world, with the help of a new editing tool called Crisper (CRISPR).

Presumably the mushroom farmers in the audience had never heard of CRISPR, but they understood its importance when Young showed a photo of actress Cameron Diaz giving the reality, Jennifer Dodna וEmmanuel Charpentier, the "breakthrough" award in November 2014, accompanied by a check for three million dollars each. And they realized the huge commercial implications when Yang showed them pictures of rotting brown mushrooms versus A. Bisophorus snowdrops that have undergone genetic engineering with the help of CRISPR. This is the same versatile mushroom variety that is responsible for more than 400,000 kilograms annually of white button mushrooms: carmini and portablo. (The University of Pennsylvania also realized the commercial implications. The day before Young's lecture, the university filed for a patent on mushroom research.)

In the short time of the three years in which Crisper's scientific story was told, more fascinating subplots were born in it than in a Dickens novel. It is a revolutionary research tool with dramatic medical implications, complicated ethical issues, an embarrassing patent dispute, and most of all, multi-billion dollar commercial implications in the fields of medicine and agriculture. The method swept the scientific community like a tornado. Academic laboratories and biotechnology companies are trying to develop innovative treatments for diseases such as sickle cell anemia and beta-thalassemia. And there are even rumors of hobbyist biotech artisans and entrepreneurs making everything from purple-furred rabbits to living, breathing genetically engineered toys, such as the miniaturized pigs recently made as pets in China. The possibility of using the CRISPR method to repair human embryos or permanently edit our DNA (a process known as human germ cell modification) has sparked heated discussions about the "improvement" of the human race and calls for a global suspension of research in this direction.

But the most profound impact of the CRISPR revolution and less publicized, is precisely in agriculture. By the fall of 2015, about 50 scientific papers had been published on the use of CRISPR to edit genes in plants, and there are plenty of signs that the USDA, one of the agencies that monitors genetically modified agricultural products, does not believe that gene-edited crops should receive the same regulatory attention as genetically engineered organisms. "Traditional" genetics (GMOs). The gap opened in the legislative door causes companies to rush and introduce genetically modified crops into the fields, and eventually into the food supply chain.

CRISPR's ability to change DNA lies in the unusual precision of the method. By using CRISPR it is possible to eliminate any gene, or with a little more effort, add any desired trait by inserting a gene in a certain place in the genome. This makes the method, according to its users, the least biologically disruptive form ever invented by humans for developing plant varieties, including the "natural" improvement methods that have been used by mankind for thousands of years. It also allows scientists to avoid in many cases controversial methods involving the introduction of DNA from other species into plants. These "transgenic" varieties, such as the company's corn and soybeans Monsanto resistant to herbicides Roundup, particularly angered GMO critics, and led to public distrust of the method. However, some scientists express optimism and believe that CRISPR crops are fundamentally different and therefore will change the tone of the debate compared to GMO foods. "The new method," he says Daniel P. Voytas, a researcher in academia and industry, "makes us rethink the definition of genetically modified organisms."

Will consumers agree? Or will they think that Crisper crops are simply the latest version of "Frankenstein food" - a genetic distortion of nature, in which foreign (and agribusiness-friendly) DNA is inserted into plants, with unexpected health and environmental consequences? Since the CRISPR method has only recently been applied to edible crops, the question has not yet come up publicly, but it will soon. Farmers like Yang's mushroom growers will be the first to have their say, probably in the next year or two.

A few minutes after Young's lecture, a scientist from the industry confronted him with the main challenge of CRISPR crops. The researcher agreed with Young that the improved mushrooms are the result of minimal interference with the DNA compared to regular GMOs. "But," said the scientist, "it is still genetic engineering, and some people will think that we are playing the role of God. How do you overcome it?" How Yang and other scientists applying these gene-editing methods to food can answer the question will determine whether CRISPR is a potentially transformative tool or one that will fail in the face of public opposition.

"Wow, that's it!"

The telltale sign of any revolutionary method is the speed with which researchers apply it to their own research. By this standard, CRISPR ranks among the most important additions to the biological toolbox in the last half century. The genetically edited mushrooms demonstrate this.

Yinong Yang, whose first name in Chinese means "also does agriculture", hadn't worked with mushrooms before 2013, but you could say he was up to the task. He was born in Huangyan, a city south of Shanghai known as the citrus capital of China, and experienced working with primitive gene-editing enzymes in the mid-90s as a doctoral student at the University of Florida and then at the University of Arkansas. He clearly remembers opening the August 17, 2012, issue of the journal Science, which contained an article that presented the work being done in Doudna's lab at UC Berkeley and Charpentier's lab, and described the potential of CRISPR's gene-editing method. "Wow," he thought. "that's it!" Within days he made plans to improve the properties of rice and potatoes with the help of gene editing. His lab published its first CRISPR paper in the summer of 2013.

He wasn't the only one. Plant researchers found great interest in CRISPR from the moment the method was published. Chinese scientists, who quickly adopted the method, stunned the agricultural community in 2014 when they showed how CRISPR could be used to develop bread wheat resistant to an old pest, the mealybug.

However, the gene editing revolution began before the appearance of CRISPR. For people like Veytas, CRISPR is just the latest chapter in a much longer scientific saga that is only now beginning to bear fruit. He first tried to edit genes in plants 15 years ago, while at the University of Iowa, using a technology known as Zinc fingers. His first gene editing company failed due to patent issues. In 2008 he moved to the University of Minnesota and in 2010 he patented, with the help of a colleague who was previously at the University of Iowa and now works at Cornell University, a system for editing genes in plants based onTALENs, another gene editing tool. In the same year, Vytas and his colleagues founded a company called today calyxt. Without CRISPR's PR, plant researchers have used TALENs to produce genetically edited plants that are already growing in fields in North and South America. Clixt, for example, has developed two strains of soy that produce a healthier oil, with levels of monounsaturated fats similar to those in olive oil or canola oil. The company also developed a potato variety to prevent the accumulation of certain sugars during storage in the refrigerator, to reduce the bitter taste associated with storage, as well as the amount of acrylamide, suspected of being carcinogenic, that is formed when potatoes are fried.

Because gene editing does not involve the introduction of any foreign genes, the US Department of Agriculture's Animal and Plant Inspection Service (APHIS) decided in 2015 that these crops do not need to be controlled like GMOs. “The U.S. Department of Agriculture has approved the cultivation of one potato variety and two soybean varieties, so this potato and one of the soybean varieties are already in the fields this year,” Veytas told me in October 2015. “They're actually treating them like normal plants, like they were created through chemical mutation or rays Gamma or any other method that does not require control through legislation. The fact that we received regulatory approval and we can move almost immediately from the greenhouse to the field is a big advantage. This allows us to significantly speed up product development."

Animal researchers have also jumped on the gene editing bandwagon. Researchers at the small biotechnology company Recombinetics, in Minnesota, genetically blocked the biological signal that controls horn growth in cows Holstein, which are the basis of the dairy industry. They did this by using gene editing to copy a mutation that occurs naturally in the hornless breed of Angus cattle. Scientists in the agricultural industry praise this application of gene editing as a more humane solution because it eliminates the need to perform a brutal procedure on Holstein cattle in which ranchers physically remove the horn buds and then cauterize the site (the procedure is done to protect the ranchers and the cows from injury). Scott Frankrog, the company's CEO, says that the development does not involve transgenes (foreign genes), but only the insertion of a few DNA letters "that fit the food we already eat." Meanwhile, Korean and Chinese scientists have teamed up to produce a pig with much more muscle mass by using gene editing to disable the activity of a gene called Myostatin.

CRISPR's speed, ease and economy are the reasons why the method has priority over TALENs. "Without a doubt," says Voytas, in the future Crisper "will be the tool of choice for editing plants." But the ambiguity in terms of patents related to the method may slow down commercial agricultural developments. The controversy stems from two institutions, the University of California and the Broad Institute (jointly run by the Massachusetts Institute of Technology and Harvard University), claiming to have invented CRISPR. DuPont recently formed a "strategic partnership" with Caribou Biosciences, a biotech company affiliated with the University of California at Berkeley, to use CRISPR's applications in agriculture, but executives at two small biotech companies told Scientific American they are wary of developing CRISPR-related products while the patent dispute remains.

But the ambiguity regarding the patent does not concern university laboratories. The story of the fungus took a decisive turn in October 2013, when David Carroll, a graduate of Penn State University, dropped by Young's lab for a visit. Carroll, who happened to be the president of the mushroom company Giorgio, wondered if the new gene editing methods could be used to improve fungi. Encouraged by CRISPR's power to produce extremely precise mutations, Young asked: "What kind of trait do you want?" Carroll suggested anti-browning, and Young immediately agreed to try.

Yang knew exactly which gene to target. Biologists previously identified a family of six genes, each of which encodes an enzyme that causes browning (the same group of genes also causes browning in apples and potatoes, and in both the genes were changed through gene editing). Four of the browning genes produce this enzyme in the fruiting body of mushrooms, and Yang thought that if he silenced one of them with a mutation, he might slow the rate of browning.

The incredible convenience of CRISPR comes from the fact that it is easy for biologists to adapt the molecular tool that produces such mutations. Like a penknife that combines a compass, scissors, and clamps, these tools excel at two tasks: framing a very specific DNA sequence and then cutting it (the clamps, or scaffolding, hold the entire structure in place during cutting). The domestication is due to a small piece of nucleic acid called Guide RNA, which is designed to be a mirror image of the DNA sequence at the target site and stick to it with the help of the unique and specific attraction forces of DNA pairs, discovered by James Watson and Francis Crick (A binds only to T and C binds only to G). If you prepare a piece of guide RNA 20 letters long, it will find its mirror image sequence in the DNA, with the precision of a GPS, within the long string of 30 million letters in the genome of the agaricus mushroom. The cutting is completed using a so-called enzymeCas9, which was originally isolated from cultures of yogurt bacteria. The enzyme is carried to the DNA on top of the guide RNA. (The full accepted term for the method, CRISPR/Cas9, has now become a misnomer. The acronym CRISPR refers to DNA segments found only in bacteria, and they were used in the discovery of the method. Today, the Cas9 protein, which carries an RNA sequence that directs it to a target, is the one that edits DNA of plants, fungi and humans, although CRISPR sequences are not involved at all.)

From the moment the gene editors cut the DNA at the desired point, they allow nature to do the dirty work of creating mutations. Every time the DNA double helix is ​​cut, the cell notices the wound and repairs it. But the repair process is imperfect, which is what makes the CRISPR method so effective at creating mutations. In the repair process, some DNA letters often fall out. Because the protein-making mechanism in the cell reads the DNA in three-letter "words", missing a few letters changes the entire text and actually paralyzes the gene, creating what is known as a shift in the reading frame. This is exactly what happened in genetically edited fungi. In Yang's study, the missing of a small number of DNA letters paralyzed one of the enzymes that promote browning, and Yang and his colleagues confirmed the existence of the mutation in the DNA test. The editing is finished. According to Young, any skilled molecular biologist can build in three days a tool to create a desired mutation to edit any gene in any creature.

This feeling also emerges from the mantra that scientists often quote about CRISPR: it is a fast, cheap and easy method. It took about two months of laboratory work to produce the non-browning mushrooms. From the look on Yang's face it was obvious that the job was routine, and ridiculously easy. And incredibly cheap. The most difficult step, preparing the guide RNA and its template, cost several hundred dollars. Several small biotech companies are now making CRISPR constructs to order to edit any gene you want. The highest cost is manpower. Xiangling Shen, a postdoctoral fellow in Yang's lab, worked on the project part-time. "If you don't factor in the manpower, it must have cost less than $10,000," Young says. In the world of agricultural biotechnology, that's small money.

But the big savings that will change the rules of the game may be in the area of ​​legal approvals. In October 2015, Young gave an informal lecture on the fungus research to federal regulators at the USDA's Animal and Plant Inspection Service, who decide whether genetically engineered crops should be subject to government regulation (i.e. whether they are considered GMOs). He left the meeting with a strong feeling that the regulators of the Ministry of Agriculture do not believe that the Crisper mushrooms will need a special or lengthy regulatory process. If this is indeed the case, this may be the most important savings for CRISPR: Veytas estimates that the regulatory approval process could cost up to $35 million and take up to five and a half years.

Another advantage of the mushroom as proof of the feasibility of Crisper in agriculture is the speed with which the mushrooms grow. From a spore to a mature mushroom takes about five weeks, and mushrooms can be grown year-round in windowless, climate-controlled facilities. On the other hand, it took months to test Clixt's genetically modified soybeans and potatoes in the field, which is why the company requested and received permission to grow soybeans in Argentina in the winter of 2014-2015. "Jump back and forth on both sides of the equator," says Voytas, "so that it is possible to sow several times a year." Clixt harvested its first GM crops in North America in October 2015.

One of the long-term concerns about genetic engineering is the unpredictable results it can cause. In the biotechnological food world, this means unexpected toxins or allergens that will make the food unhealthy (a concern that has never been proven in GMO food) or a genetically modified crop that will suddenly get out of control and destroy the local ecology. Crisper makes even people like John Peccia Think about unexpected financial consequences. Peccia, one of two professors specializing in fungi at Penn State University, spends a lot of time in a low brick building on the outskirts of campus that houses the only US center for academic fungal research. In the spring of 2015, Peccia took some of Young's mushroom culture and grew the first crop of genetically edited mushrooms. Standing outside a room where a steaming, steaming mixture of mushroom fertilizer is being cooked at 80 degrees Celsius, he says a mushroom with a longer shelf life could result in lower demand from stores and also allow for unexpected competition. "It will be possible to open the borders to import foreign mushrooms," he adds, "so it is a double-edged sword."

In the exhausting path of genetically modified food to the market, here is another reason for reflection. No one knows what the genetically edited mushroom tastes like. They were steamed and boiled, but not for edible purposes. Every mushroom that had been created so far was destroyed after Yang performed the browning test. Once the proof of feasibility is achieved, says Peccia, "we just steam them to death."

Changes without transgenes

Will the public steam, fry or in any way accept genetically modified food for the kitchen and from there for the plate? This may be the central question in the most fascinating chapter of the food and Crisper story, which coincides with a crucial juncture in the 30-year-old debate about genetically engineered food.

When Yang described his mushroom project to Pennsylvania farmers and USDA officials in October 2015, he used a key phrase to describe the process: "genetic modification without transgenes." This is a premeditated attempt to distinguish between the new and precise methods for editing genes such as CRISPR, and between older agricultural biotechnology in which foreign (transgenic) DNA segments have been inserted into plants. For Yang and many others, the subtle wording is important to rewriting the GMO debate. Indeed, the acronym GEO (for genetically modified organisms), began to emerge as an alternative to GMO or GM.

New technologies like CRISPR are forcing some governments to reconsider the definition of a genetically modified organism.

The rebranding is not only semantic, but philosophical as well, and is being revealed due to the changes the Obama administration is making in the way the government evaluates genetically engineered crops and food. This control process, which has not been updated since 1992, defines the roles of the Department of Agriculture, the Food and Drug Administration and the US Environmental Protection Agency. The power of CRISPR increases the urgency of renewed regulatory thinking, and scientists use the opportunity to ask again a very old question: what exactly does "genetically engineered" mean? Veitas, whose list of publications and patents on genetically edited food makes him a sort of editor-in-chief of small companies specializing in agricultural biotechnology in the US, replied with a dark chuckle: "The term GM is definitely problematic."

What is so problematic about it? Most critics of biotechnological food claim that any form of genetic modification is what it is, genetic modification, which carries with it the possibility of unplanned mutations or changes that could endanger public health or the environment. Scientists like Veitas and Young answer that all forms of plant breeding, which began with the creation of bread wheat by Neolithic farmers 3,000 years ago, involve genetic changes and that even traditional breeding methods have a biological effect. They create, as Young puts it, "enormous" genetic disorders. (Nina Federoff, a plant biologist and former president of the American Association for the Advancement of Science, referred to the domesticated versions of bread wheat, created by traditional hybrids, as "genetic freaks.")

Before the era of recombinant DNA in the 70s, which enabled the creation of the first generation of agricultural biotechnology, plant breeders often used aggressive methods (X-rays, gamma rays, or powerful chemicals) to alter the DNA of plants. . Despite the unsophisticated approach, some of these random, man-made mutations have altered genes in a way that leads to desirable agricultural traits, such as higher yields, better-shaped fruit, or the ability to grow in harsh conditions such as drought. These beneficial mutations were combined with desirable traits in other varieties, but only through hybridization, i.e. pairing of plants. This type of crossbreeding takes a long time (usually five to ten years), but at least it is "natural".

But it is also very harmful. Whenever DNA from two different individuals comes into contact during reproduction, whether in humans or in plants, the DNA gets mixed up in a process known as chromosome rearrangement. When plant breeders focus on a desired trait, spontaneous mutations can happen every generation, and millions of DNA base pairs can be passed from one specification to another. It is indeed a natural process, but it is also "one big mess," according to Vytas. "In this process, you don't move just one gene," he says. "Often a fairly large piece of DNA is moved from the wild strain." Moreover, during the breeding process the desired trait often brings with it an unwanted trait on the same piece of DNA. This "drag in the grip" can even harm a plant that has undergone natural improvement. Based on some recent findings regarding the genetics of rice plants, some biologists speculate that the domestication process accidentally introduced harmful "silent" mutations in addition to the obvious beneficial properties.

Although CRISPR is more accurate than traditional enhancement, the method is not immune to errors. The precision cutting tool sometimes cuts in areas that were not planned in advance, and the prevalence of this "off-target" cutting has raised safety concerns (and is also the main reason why gene editing in human eggs and sperm cells is still considered unsafe and unethical). Jennifer Kuzma, a policy analyst at the University of North Carolina who has followed the science and politics of GMO agriculture since its inception, says: "This precision has benefits, but it doesn't necessarily reduce risk." Cutting in the wrong place "could pave a new path to danger." Feng Zhang From the Broad Institute (the owner of the patent that is now in dispute) published several innovations included in the CRISPR system that improve specificity and reduce cutting in wrong places.

CRISPR's ease of use and savings have also allowed academic labs and small biotech companies to return to the game that until now was run by large agricultural businesses. Only companies with deep pockets could initially afford to meet the expensive challenge of issuing permits, and today almost every change in crops created through genetic engineering is done to increase the economics of food production for the farmers or companies, and one is if it is about increasing the yield of field crops resistant to herbicides of Monsanto or in resistance to transport of The genetically engineered tomatoes The unlucky ones of the Kalgin company. These changes in crop plants were more tempting to the agricultural companies than to the consumers, and they did not concentrate on the quality of the food. As a group of agricultural policy experts at the University of California at Davis recently explained, "The multinational corporations that have dominated the field for the past decade and a half do not have a bright past in terms of innovation, other than traits that confer resistance to insecticides or herbicides."

The new players bring a different kind of innovation to agriculture. Veitas, for example, claims that the precision of gene editing allows biotechnologists to focus on consumers by creating healthier and safer food. Veitas and his colleague Caisia ​​Gao from the Chinese Academy of Sciences point out that plants have many "anti-nutrients": harmful substances designed for self-defense or actual toxins that can be removed through gene editing to improve nutrition and taste properties. Clixt's genetically modified potato, for example, reduces the bitter taste characteristic associated with cold storage of the pomod.

But Vytas does not stop there. He believes that Clixt soybeans will be sold to farmers as non-GMO food because, unlike 90% of soybeans grown in the US, the genetically edited varieties do not contain transgenes. "A lot of people don't want GM products," he says. "With the help of our product, we may be able to make non-GM soy oil and soy bran."

Like any new and powerful technology, CRISPR inspires some agricultural visionaries who imagine future scenarios almost out of the realm of science fiction - scenarios that are already finding their way into the scientific literature. Michael Palmgren, a plant biologist at the University of Copenhagen, believes that scientists can use the new gene-editing methods to return plants to their wild state, that is, to revive traits that have been lost through generations of agricultural improvement. Some economically important food crops, notably rice, wheat, oranges and bananas, are particularly susceptible to pests. Restoring lost genes may increase disease resistance. The goal, as Palmgren and his Danish colleagues recently noted, is to "correct the unexpected results of plant breeding."

Attempts to restore the state of the bar are already underway, but with a small change. Instead of reintroducing lost wild traits to domesticated varieties, Voytas says his lab at the University of Minnesota is trying to do what he calls "molecular domestication": transferring agriculturally desirable genes from existing varieties back into hardier and more adaptable wild varieties, such as early forms of corn and potatoes. "Usually it's a handful of critical changes that occurred - five, six or seven genes - that allowed a grassy variety to become desirable, such as changes in the size of the fruit or the number of corn cobs, things like that," says Veitas. Instead of crossing the wild strains with domesticated strains, a hybridization process that takes ten years, he says, "Maybe we can just take care of these genes and domesticate the wild strains."

There are early signs that gene editing, including CRISPR, may benefit from a faster approval process. So far, it seems that the American regulators believe that at least some of the genetically edited crops are different from transgenic GMO crops. When Clixt first approached the USDA and asked whether genetically edited potatoes needed regulatory review, it took federal officials a full year to decide, on August 14, that gene editing did not require special considerations. When the company approached the Ministry of Agriculture again last summer with the genetically modified soybeans, it took government officials only two months to reach a similar conclusion. In the eyes of companies, this indicates that the US authorities believe that the new methods are fundamentally different from transgenic methods. In the eyes of critics, this indicates a regulatory loophole that the companies are exploiting. Young's mushrooms may be the first crisper food to be discussed at the USDA.

And new technologies like CRISPR are forcing some governments to reconsider the definition of GMO. In November 2015, the Swedish Ministry of Agriculture ruled that some of the mutations in the plants cultivated using the CRISPR method do not meet the European Union's definition of GMO, and Argentina also concluded that some of the genetically edited plants are not included in the GMO regulations. The European Union, which has historically opposed genetically modified plants, is reconsidering its position in the face of the new genetic editing methods, but its legal analysis, which was supposed to be published by the end of March 2016 at the earliest, is delayed. Although there is not much in common between the researchers and the opponents, Veitas and others have proposed one possible compromise: gene editing that causes mutation, or gene silencing, would be considered like more traditional forms of plant breeding (such as the use of X-rays to produce mutations), while gene editing that introduces DNA A new will undergo regulatory control on a case-by-case basis.

The day of genetically edited crops may be quite close. Veitas estimates that Clixt will have a "small commercial launch" of its soybeans by 2017 or 2018. "It will take some time to have enough seeds for, say, two million hectares," he says. "But we are moving forward with all our might."

How will the public react? Kuzma predicts that people who previously opposed genetic modification will not be drinking from the Crisper any time soon. "The public that opposed the first generation of GMO food is not expected to embrace the second generation of genetic engineering, just because we change a little bit of DNA," she says. "They will bundle everything together with GMOs." Kuzma is more concerned about the need to overhaul the entire regulatory structure and include more voices in the control process, at a time when more and more genetically edited foods are making their way to the market.

And what about the mushrooms? Beyond the polite applause at the end of Yinong Yang's lecture, the reaction of the mushroom farmers is unclear. Young also understood this when he told the farmers: "Whether it can be commercialized is up to you." For now, the non-browning mushrooms are just a lab project, a proof of feasibility. If farmers are not convinced of the value of the mushrooms or fear that consumers will shy away from them, the edited mushrooms may not see the light of day. That's usually a good thing for the mushrooms, which grow in the dark, but a bad sign for this breakthrough technology.