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A new toolbox for better crops Plant scientists are using CRISPR gene editing to make higher-quality, more sustainable agriculture products, but consumer acceptance is not guaranteed MELODY M. BOMGARDNER, C&EN WEST COAST
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CREDIT: C&EN/SHUTTERSTOCK
ometime around 2020, a new corn variety will mark a huge leap in how humans design agricultural crops. It will be the first commercialized, gene-edited plant altered using CRISPR/Cas9 technology. But don’t be surprised if the corn debuts without much hype. It is a starchy or “waxy” corn that is not much different from varieties already on the market. When the seed firm DuPont Pioneer first announced the new corn in early 2016, few people paid attention. Pharmaceutical companies using CRISPR for new drugs got the headlines instead. But people should notice DuPont’s waxy corn because using CRISPR—an acronym for clustered regularly interspaced short palindromic repeats—to delete or alter traits in plants is changing the world of plant breeding, scientists say. Moreover, the technique’s application in agriculture is likely to reach the public years before CRISPR-aided drugs hit the market. Until CRISPR tools were developed, the process of finding useful traits and getting them into reliable, productive plants took many years. It involved a lot of steps and was plagued by randomness. “Now, because of basic research in the lab and in the field, we can go straight after the traits we want,” says Zachary Lippman, professor of biological sciences at Cold Spring Harbor Laboratory. CRISPR has been transformative, Lippman says. “It’s basically a freight train that’s not going to stop.” Using CRISPR to add—or remove—a plant trait is faster, more precise, easier, and in most cases cheaper than either traditional breeding techniques or older genetic engineering methods. Although scientists can use CRISPR to add genes from other species to a plant, many labs are working to exploit the vast diversity of genes that exists within a plant species. In fact, enhancing many of the most valued traits in agriculture doesn’t require adding DNA from other species. Gene-edited crops have the potential to revive some of the early promise that genetic engineering has not fulfilled, such as making plants that are higher yielding, drought tolerant, disease resistant, more nutritious, or just better tasting. In addition, CRISPR can efficiently improve not just row crops such as corn but also fruits
and vegetables, ornamentals, and staple crops such as cassava. Proponents hope consumers will embrace gene-edited crops in a way that they did not accept genetically engineered ones, especially because they needn’t involve the introduction of genes from other species—a process that gave rise to the specter of Frankenfood. But it’s not clear how consumers will react or if gene editing will result in traits that consumers value. And the potential commercial uses of CRISPR may narrow if agriculture agencies in the U.S. and Europe decide to regulate gene-edited crops in the same way they do genetically engineered crops. DuPont Pioneer expects the U.S. to treat its gene-edited waxy corn like a conventional crop because it does not contain any foreign genes, according to Neal Gutterson, the company’s vice president of R&D. In fact, the waxy trait already exists in some corn varieties. It gives the kernels a starch content of more than 97% amylopectin, compared with 75% amylopectin in regular feed corn. The rest of the kernel is amylose. Amylopectin is more soluble than amylose, making starch from waxy corn a better choice for paper adhesives and food thickeners. Like most of today’s crops, DuPont’s current waxy corn varieties are the result of decades of effort by plant breeders using conventional breeding techniques. Breeders identify new traits by examining unusual, or mutant, plants. Over many generations of breeding, they work to get a desired trait into high-performing (elite) varieties that lack the trait. They begin with a first-generation cross, or hybrid, of a mutant and an elite plant and then breed
In brief The first CRISPR gene-edited crops are coming. A new waxy corn variety from DuPont Pioneer will hit the market in about three years. And given the speed, ease, and wide use of CRISPR gene editing, many other crops are sure to follow. Compared with traditional breeding and older genetic engineering techniques, CRISPR is much more precise: A gene-edited plant with a target trait can be produced in one generation. In the pages that follow, C&EN explores how people are using CRISPR to develop new varieties of corn, tomatoes, and cotton. Yet despite the clear technological advantages of the strategy, proponents don’t know how it will be regulated or if consumers will embrace it.
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several generations of hybrids with the elite hybrid, to get the new corn to farmers. one at a time. And it’s not hard to do.” parent in a process called backcrossing. Waxy corn was an ideal variety on Sederoff’s lab is studying ways to inThey aim to achieve a plant that best apwhich to try CRISPR for a first commercial crease the amount of oil produced by oilproximates the elite version with the new product, Gutterson says. It has a trait that seeds such as canola and the industrial crop trait. has been long marketed and is familiar to camelina. Her team is looking for genes But it’s tough to grab only the desired farmers. that control how a plant transports sugar trait from a mutant and make a clean getAnother reason was that plant scientists or regulates the amount of sugar that goes away. DuPont’s plant scientists found that understand the corn genome and the waxy out of its stem and into the seed, where it the waxy trait came with some genetic trait in particular. “You really have to unis converted into fatty acids. “Can we make baggage; even after backcrossing, the waxy derstand the gene for the trait, the genome, more seeds? Can we change the composicorn plant did not offer the same yield as and the effect of the edit,” Gutterson says. tion or size of the seeds?” she asks. elite versions without the trait. The disap“Many versions of this gene exist in nature. In one set of experiments, Sederoff pointing outcome is common enough that It made it easy for us to get exactly the used CRISPR to place a gene that makes it has its own term: yield drag. property we want.” tomatoes sweet into an oilseed plant. Seed Because the waxy trait is native to cerAccording to plant scientists, better unyield doubled. She reports it took less than tain corn plants, DuPont did not have to derstanding of a species’ genome, including two years, compared with the 10 years that rely on the genetic engineering techniques the identity of genes that code for desired older techniques would require. In the long that breeders have used to make herbitraits, is the main hurdle to widespread run, researchers might find and use native cide-tolerant and insect-resistant oilseed genes that work like the corn plants. Those commonly one taken from the tomato to planted crops contain DNA from CRISPR technology employs a guide RNA to direct the Cas9 create a higher-yielding crop that other species. enzyme (light blue) to a target DNA sequence. Once there, isn’t transgenic. In addition to giving some Cas9 will bind when it finds a protospacer-adjacent motif Cold Spring Harbor’s Lippman consumers pause, that process sequence (red) in the DNA and cut both strands, priming is also working with tomatoes. does not precisely place the DNA the gene sequence for editing. His team is looking for the genes into the host plant. So researchers that control how many, when, and must raise hundreds or thousands where flowers—and thus tomaof modified plants to find the best toes—are produced on plants. Cas9 ones with the desired trait and That means understanding what work to get that trait into each happens in the stem cells that Genomic DNA elite variety. Finally, plants modiproduce flower branches, called fied with traditional genetic engiinflorescences. Target sequence neering need regulatory approval In the past, breeders had trouin the U.S. and other countries ble fine-tuning the amount and before they can be marketed. pattern of inflorescences. The Instead, DuPont plant scienproblem, Lippman discovered, is tists used CRISPR to zero in on, that two traits that arose during Guide RNA and partially knock out, a gene for decades of domestication and an enzyme that produces amycrop improvement combined to lose. By editing the gene directly, thwart the altering of flower prothey created a waxy version of duction via additional breeding. the elite corn without yield drag or foreign use of gene editing. Researchers have had One of the traits helped the plant support DNA. access to the full corn genome only since heavier fruit; the other eliminated a joint Plant scientists who adopt gene editing 2010, and they are still sequencing a numon the fruit stem to prevent tomatoes from may still need to breed, measure, and obber of important corn varieties. falling off before harvesting. serve because traits might not work well “Plants—like animals—have lots of With CRISPR, Lippman notes, what was together or bring a meaningful benefit. “It’s genes, most of which we don’t understand,” done can be undone. “We have ways now to not a panacea,” Lippman says, “but it is one notes Heike Sederoff, professor of systems use gene editing to separately modify fruit of the most powerful tools to come around, and synthetic biology at North Carolina size and weight, the branches that make ever.” State University. “We don’t know what they flowers, and the amount of flowers, as well DuPont was an early adopter of CRISPR do or why they are there or how they got as the architecture of a plant from a comtechnologies, before Monsanto and other there.” pact bush to one that keeps growing.” seed industry rivals. In 2015, the company But here, too, CRISPR easily beats out A different breeding mistake may be to signed technology license deals with Vilcompeting techniques. To figure out the blame for modern tomato varieties’ lack of nius University and Caribou Biosciences. function of one of the 20,000 to 30,000 flavor and aroma. Research shows that as Caribou was founded by CRISPR research genes in a plant, scientists either knock out breeders sought traits for productivity, unipioneer Jennifer Doudna of the University the gene or dial up its impact by adding cop- formity, and harvest-ability, the tastier traits of California, Berkeley. ies. “We used to use viruses or bacteria that were inadvertently lost. Wild tomatoes and Gutterson says his team started work insert DNA, but the targeting part is really heirloom varieties still carry those genes. on the new waxy corn in early 2015. “One difficult,” Sederoff says. “Now let’s breed them in or edit them to observation or lesson we have with our first “That’s where CRISPR helps us. It albring back a better-flavored tomato, which product is that the reduced time to market lows us to very specifically target a gene is what everybody asks for all of the time,” is significant,” he says. It will take less than and either take it out or modify it. We can Lippman says. five years, compared with about eight for a study any gene, and we can do more than Cotton growers are also excited about
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CREDIT: ADAPTED FROM ORIGENE TECHNOLOGIES
Targeted change
the quality improvement that CRISPR gene editing could bring. “Cotton is a small-acreage crop compared with corn and soy,” explains Kater D. Hake, vice president of agricultural and environmental research at Cotton Inc., a promotion organization supported by cotton farmers. “With the regulatory cost associated with traditional biotechnology, cotton has been off the radar except for extremely high-value traits such as insect and weed control.” Researchers are probing the cotton genome, which was first sequenced in 2015, to find genes that control the shape, structure, length, and strength of cotton fibers. “It’s a sustainability story,” Hake says. “When you push cotton quality up, you can make stronger, finer yarns so garments require less total mass of cotton and are more durable.” Indeed, researchers have no shortage of ideas for how to use CRISPR to make higher-quality, more sustainable crops that consumers may desire. But to date, most of the work has been to prove the concept. It’s not yet clear which innovations will actually reach the market. One concern is that smaller seed firms and research organizations aren’t geared up to develop and commercialize crops with new traits; they ceded most of that ground to agriculture giants such as DuPont decades ago. Benson Hill Biosystems, a St. Louis-based start-up, is working with small seed companies and academic researchers to help them pursue crop improvement projects using its data-driven genomics platform. For example, the firm is working with the family-owned seed firm Beck’s Hybrids and potato experts at J.R. Simplot Company to bring more R&D power in-house. “We believe DuPont and Monsanto will play a decreasing role relative to innovation across the industry,” Benson Hill Chief Executive Officer Matthew Crisp asserts. “It will be like the shift in big pharma 10–15 years ago when early-stage discovery went to smaller players.” Crisp says CRISPR gene editing and genomic data tools will level the playing field for new trait introductions. Another constraint is that a few organizations control important patents for CRISPR, some of which have been the subject of lawsuits. So scientists at Benson Hill are working on a way to replace Cas9, the enzyme that cuts the DNA. Crisp calls the work “CRISPR 2.0” and says he expects the tools to be even more efficient—and easier to access—than current ones.
These four foods are ripe for CRISPR gene editing Agriculture observers see tomatoes, mushrooms, wheat, and corn among the first to benefit from the hot new technique
A better-tasting tomato “Deterioration in flavor quality of the modern commercial tomato relative to heirloom varieties is a major cause of consumer complaint,” an international team of geneticists dryly note in a recent paper. The head of the team, Harry J. Klee of the Plant Innovation Center at the University of Florida, and coauthors examined 398 modern, heirloom, and wild tomatoes to identify missing flavor components. They found that 13 important flavor volatiles, including guaiacol and methyl salicylate, were significantly reduced in modern varieties and identified the locations of genes responsible for making the chemicals. In addition, they found that efforts to breed larger fruit had resulted in lower sugar content. The team says the research points to genes that can improve flavor quality in tomatoes and other fruit crops (Science 2017, DOI: 10.1126/science.aal1556). While other groups have shown that CRISPR can be used to edit genes in tomatoes, it has not yet been used to add a missing gene.
Nonbrowning mushrooms White button mushrooms, once they are bruised or sliced, don’t stay white for long. The culprit behind mushroom browning is the pigment melanin. Pennsylvania State University associate professor Yinong Yang traced the melanin production to a specific polyphenol oxidase gene in the mushroom and used CRISPR/Cas9 to make a small deletion in the gene. The result was nonbrowning mushrooms. But Yang is famous in the biotechnology community for what happened next: In October 2015, he wrote a letter to the U.S. Department of Agriculture asking it to confirm that his mushroom, which does not contain foreign genes, would not be regulated. Five months later, USDA wrote back with confirmation. The ruling opened the door for many other crops developed using CRISPR/Cas9 technology. Yang has filed a provisional patent application for the mushroom.
Mildew-resistant wheat Many important agriculture crops are polyploids, meaning they have several copies of genes that may not all be identical. Wheat has three copies of most of its genes, including the gene that makes it susceptible to powdery mildew, one of the world’s most destructive plant pathogens. The crop’s complicated genome hinders efforts to create a resistant plant using traditional breeding. Yanpeng Wang of the Institute of Genetics & Developmental Biology at the Chinese Academy of Sciences and coauthors used CRISPR/Cas9 as well as transcription activator-like effector nuclease (TALEN) editing techniques to deactivate the susceptibility genes and turn up the plant’s defenses against powdery mildew (Nat. Biotechnol. 2014, DOI: 10.1038/nbt.2969).
Drought-resistant corn Researchers at DuPont Pioneer built on earlier studies that found transgenic corn plants that overexpress a gene called ARGOS8 produce more grain under drought conditions than nontransgenic plants do. The gene reduces the plant’s sensitivity to ethylene, an important plant stress hormone. The team used a gene promoter for ARGOS8 from a native corn variety and inserted it via CRISPR into nontransgenic plants. The result was the plants produced more bushels per hectare during drought conditions and did not lose yield under well-watered conditions (Plant Biotechnol. J. 2017, DOI: 10.1111/pbi.12603). JUNE 12, 2017 | CEN.ACS.ORG | C&EN
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The Lippman group uses CRISPR gene editing to alter the number and branching pattern of flowers that become tomato fruit. you put in,” says Gregory Jaffe, director of the biotechnology project at the consumer advocacy group Center for Science in the Public Interest. “Clearly, one aspect of doing risk assessment is that how you put the trait in could inform risk assessment.” Using a Brazil nut gene to improve disease resistance, for example, could introduce a nonnative protein that may be allergenic, Jaffe points out. Jaffe and others say regulatory changes and the new editing technologies could blur the line between what is and is not a genetically modified organism (GMO). Currently, food containing genetically modified ingredients must carry a label. It’s not clear if CRISPR-edited products will also require a label.
That’s one reason why Jaffe has proposed a registry for both the public and the food industry to track what crops come from gene editing. “It’s important not to make the kinds of mistakes that were made with GMO crops,” Jaffe says. “We should start with more transparency in the food chain.” Benson Hill’s Crisp agrees that the industry must be more transparent and do a better job at outreach. “We need to ensure that consumers are informed about the benefits and not inundated with misinformation or a lack of information.” Food shoppers will likely be won over with gene-edited products that directly benefit them, Jaffe predicts. And he’s already got something in mind: “I would like a better-tasting tomato.” ◾
CREDIT: ZACHARY LIPPMAN
Researchers at the University of California, Berkeley, are also developing Cas9 alternatives. But as CRISPR technology advances, questions persist about government regulation and consumer acceptance. Today, companies that wish to market a gene-edited plant can ask the U.S. Department of Agriculture whether their product will require regulatory review. So far, for plants that do not contain foreign genes, USDA has responded that it does not have the authority to regulate. Transgenic plants, in contrast, are regulated because they contain genes from other species or from a vector organism that may introduce a plant pest into the environment. That regulatory framework, which was set up in 1987, is undergoing a comprehensive review; USDA is accepting comments through June 19 about how it should assess risk in modified crops. In addition, other countries may write different, more onerous rules. Many researchers share the view that regulators should focus on whether added or altered traits pose a foreseeable risk and not on the process used to get the trait into the plant. “I propose doing regulation based on the phenotype—the specific characteristics
