Agriculture might not strike you as humanity’s first scientific endeavor. Familiar images of farmers tilling their fields or cowboys tending to their cattle evoke a nostalgia at odds with the test tubes and lab coats that have come to signify modern science. Yet agriculture represents one of the first successful applications of science to a problem that has dogged human civilization since its inception: how to feed many people without year-round hunting and gathering.

Just as the practice of agriculture has evolved over thousands of years, so has our understanding of its biological underpinnings. The emergence of modern genetics in the last century has given way to more efficient manipulation of crop traits during selective breeding, spurring the development of everything from the Honeycrisp apple to genetically modified (GM) insect-resistant corn. Largely thanks to the dominance of a handful of GM cash crops on American farms, the public has become increasingly wary of the role of biotechnology in agriculture, putting the long-symbiotic relationship between science and farming up in the air.

Recent discoveries in molecular biology, led by UC Berkeley professor Jennifer Doudna and her team, have the potential to revolutionize agriculture yet again. The Doudna lab’s series of landmark findings about the CRISPR/Cas9 system of bacterial immunity (“Germ Warfare”, BSR Fall 2012) have given way to what has been dubbed by some the “CRISPR craze”–an explosion of scientific projects worldwide that have co-opted this system to precisely manipulate genes in any organism, cheaply and efficiently. The controversy over GM food is not about to disappear, but with CRISPR/Cas9, a new era of genetic engineering, and indeed plant breeding, may now be upon us.

Attracting a target audience: farmers

For thousands of years, farmers successfully bred crops with ideal traits, despite lacking any insight into how or why these traits were inherited. Both natural breeding that occurs in the wild, and selective breeding that is firmly guided by human hands, involve the transmission, intermingling, and mutation of genes across generations. Unfortunately, selective breeding relies on the probabilistic shuffling of genes that occurs when two parental strains reproduce–a roll of the dice for each trait, in each newborn seed, with no guarantees.

In the early 20th century, scientists learned that genes, the purported carriers of traits, could be randomly modified, or mutagenized, with radiation or certain chemicals (most commonly ethylmethane sulfonate, or EMS). Mutagenesis increased the diversity of traits that could be obtained, but the approach was still a gamble. In 1953, the dual discoveries of the structure of DNA (the double helix), and the function of DNA (as the molecule of heredity), provided a new explanation for how genes influenced traits (see “Toolbox”), but the tools available for genetic modification remained crude.

Judith Owiti, a post-doctoral fellow in the Lemaux Lab, operates a modern biolistic device, or "gene gun." Credit: Angela Kaczmarczyk

It took 20 more years for these tools to be significantly sharpened. Through the study of bacteria and viruses, scientists learned how to package specific genes within bacterial or viral DNA, creating recombinant DNA (rDNA) that could transport those genes into other organisms. In 1983, two universities and two companies simultaneously showed that plants could be “transformed,” or made transgenic (containing genes from a different species), using rDNA. Other researchers developed a method for transforming plant cells without using rDNA, using a “gene gun”–a modified ballistic device–to blast cells with DNA-coated metal particles. These tools paved the way for targeted engineering of GM crops.

It took a full decade for the earliest GM crops to gain approval for commercialization. The first GM crop to be sold in the US was the “Flavr Savr” tomato and was released in 1994. The Flavr Savr was designed to ripen slowly and retain its firmness, traits that would aid its sale in supermarket aisles. This early GM tomato not only failed to meet expectations, but also tasted bland; it was pulled from the market in 1998.

Meanwhile, Monsanto Company, an American chemical manufacturer, took a different approach by developing GM crops that would appeal to farmers. Monsanto was one of the four entities to have initially transformed plants in 1983 and already had a relationship with farmers as the seller of the popular weed-killing herbicide Roundup. The company knew that cash crops, such as corn and soy, could benefit from additional protection from weeds and pests.

Monsanto thus devised two strategies using rDNA that would appeal to farmers: a gene insertion (known as Roundup Ready) that protected crops from the plant growth-inhibiting effects of Roundup; and another gene insertion (known as Bt) providing crops with the same natural pesticide that was regularly sprayed on both conventional and organic crops, a protein found in the “Bt” bacterium, Bacillus thuringiensis.

Roundup Ready and Bt corn and soy seeds began to hit American seed markets in 1996, and were an immediate success with farmers. Within a decade, the majority of corn and soy grown in the US came from Monsanto’s GM seeds. Over the course of that decade, however, the allure of GM crops was not destined to last.

European Resistance

In 1998, just as the first American Bt soy harvests were being shipped into Europe, the European Union placed a moratorium on GM crop and seed importation. The move suggested doubt over the safety of GM crops, despite these crops having already gained approval from the US government. Indeed, Monsanto’s Bt crops could spare European farms from damages caused by a problematic insect, the European corn borer. Why pass up such a promising technology?

UC Berkeley professor and plant geneticist Peggy Lemaux had been working on GM crops for nearly a decade at that point, and she has noticed over the course of her career that anti-GM sentiment rarely bears any relation to the technology itself. “In my opinion, it really doesn’t have to do with the genes that are put in, or how they’re put in. It has to do with who’s doing it, who’s controlling the food supply,” she says. In the case of early GM seeds, the ‘who’ was US-based Monsanto, the largest purveyor of GM crops in the world. Bayer, a European chemical company, wouldn’t officially release its own GM crops until later in the 2000s, and Monsanto was then, and still is, one of Bayer’s major trans-Atlantic competitors.

UC Berkeley genomics professor Michael Eisen agrees with Lemaux’s assessment. “The anti-GMO thing in Europe… was driven by the fact that Americans were making all the GM crops, so it was a competitive advantage for their seed makers not to have to compete against these things.” Indeed, the World Trade Organization admonished the EU, calling the moratorium a violation of international trade laws–an indication that economics, not science, may have driven the move.

The EU lifted the moratorium in 2004 but replaced it with new regulations for GM crops, indicative of continued skepticism. Foods containing more than 0.9 percent GM ingredients were required to be labeled as such, and some EU member countries even enacted local bans on GM crops.

Fear spreads to the US

As the American public became aware of Europe’s hesitation to embrace GM crops, the technology behind GM crops–already decades old–began to suffer an image crisis. The nickname for GM foods, “Frankenfoods,” coined in the early 1990s to describe Monsanto’s GM cash crops, had secured its place in the public lexicon by 2000. Food activist groups began sounding alarms over GM food safety, even as multiple independent scientific review committees reinforced earlier assessments that GM food posed no greater risk to humans than conventional food. Various communities around the US floated local initiatives to label GM foods, “likely patterning what they were proposing based on the way Europeans were doing it,” according to Lemaux.

Much of the protest surrounding potentially beneficial GM crops centers around corporate control of the food supply rather than the technical process of genetic modification. This sign was seen at a 2013 March Against Monsanto in San Francisco.

Eisen, echoing a common sentiment amongst scientists about the vilification of GM, thinks that this fear stems from a lack of understanding about genetics and biology in general. “If people don’t understand what they’re talking about, they don’t understand why [genetic engineering] doesn’t alarm molecular biologists,” he says. Though GM techniques were already a staple in biology and in the production of insulin and cheese, the most visible examples were Monsanto’s GM corn and soy, which consumers rarely interacted with directly. “The real problem with these crops is not that they’re not useful, it’s that they are useful to farmers. [GM crops] don’t have a positive impact on consumers that they [the consumers] can see,” says Eisen.

In 2009, the blossoming organic movement in the US chose to ban GM technology in all its certified crops. Organic crops were grown using defined “natural” techniques, but a number of organic crop varieties had been bred using radiation or chemical-based mutagenesis. UC Berkeley professor of plant biology Brian Staskawicz, an expert on plant resistance to pathogens, finds this hypocrisy frustrating. “People don’t understand that you can do an experiment where you use EMS mutagenesis and blast the heck out of [a crop], and sell it as organic food.” Though mutagenesis breeding introduced more genetic modifications than the insertion of individual new genes with rDNA or the gene gun, it had the luxury of not being associated with Monsanto.

New GM crops stall

For academics like Lemaux, the story of misplaced anti-GM sentiment was all too familiar. Lemaux developed GM crops to improve sustainability, but each of her sustainable crop varieties hit a brick wall when she tried to commercialize them. “I’ve been in this field for over 20 years. Actually in the mid-‘90s, [this controversy] was going on in Europe. After a few years, I thought, ‘Oh, the US had been through its hard period, we’ll move on.’ Then the controversies came back here and they’ve never gone away." The collateral damage of the anti-GM movement’s war against Monsanto included over a decade of Lemaux’s work.

So, what exactly happened to each of Lemaux’s GM crop projects? They ended up in the basement of Koshland Hall, seeds safely stored in the hopes of one day being commercialized. Lemaux quickly rattled off three examples. “We created a barley variety that was fast-germinating. It would’ve saved millions of dollars for the brewing industries, but it didn’t go anywhere. We developed hypoallergenic wheat, which was less allergenic, so more people could eat [wheat]. We’ve also worked on genetic strategies to prevent pre-harvest sprouting, which causes millions of dollars a year in losses of wheat and similar grains, a problem that is likely to increase with global climate change. That didn’t go anywhere, except in China, where it is being commercialized by Chinese colleagues.”

Peggy Lemaux, with the seeds for her lab's hypoallergenic wheat variety. Without corporate backing, many of the Lemaux Lab's transgenic crops have been relegated to storage in the basement of Koshland Hall. Credit: Angela Kaczmarczyk.

These were but three of her lab’s inventions, all designed to improve agriculture and food, but what could have been a war over regulation of agrobusiness had become a war over biotechnology in agriculture. A major American brewing company had expressed interest in the Lemaux Lab’s proposed barley variety, but by the time the lab had developed and tested their crop, the company “didn’t want anything to do with it,” Lemaux says, due to the increasingly GM-wary public. Her hypoallergenic wheat variety had all the qualities of a sure-shot GM success story. “When people find out about it, they say, ‘Oh! Where is it? I’d like to have it,’” says Lemaux, “but it is too expensive for academics to develop. It needs to be picked up by a company.”

The Staskawicz lab ran into a similar complication when they developed a GM tomato containing a gene from peppers, which would protect Florida’s vulnerable tomato crop from bacterial spot disease. Hopes for commercializing the GM tomato were high. Given that the inserted gene was already found in peppers, “if you’ve eaten Mexican food, you’ve already eaten this protein,” Staskawicz says. He expected this would allay consumer’s concerns about mixing genes from unrelated species (peppers and tomatoes are both members of the solanaceous family of plants). Furthermore, his GM tomato “has really effective field resistance,” potentially making it an easy sell for Florida’s tomato farmers, who were struggling to control the disease by spraying their fields “blue, with copper,” according to Staskawicz. Despite initially proving its worth in 1999, the Staskawicz Lab’s GM tomato has yet to reach commercial markets.

Though genetic engineering had spawned numerous new crops that could help business, consumers, and the environment, its most visible effect on agriculture was the streamlining of cash crop farming, a change that most of the public did not find heartwarming. Ten years into the 21st century, Monsanto’s inventions from the ‘90s continued to hog the GM crop spotlight, leaving little room for scientists like Lemaux or Staskawicz to make the case for their inventions. With negative public opinion nipping GM agriculture in the bud, many projects were stranded between invention and commercialization.

A field in limbo

So what can be done to improve the chances of new GM crops to win over a skeptical public? According to Eisen, "We're stuck in this bad spot, where you have people who are responding negatively to GMOs that already exist, and people who are responding positively to GMOs in general." A public that has little interest in the underlying biology of agriculture might be more easily convinced of GM food’s utility by tangible examples that help people or the environment. “Available GMOs do not manifest the potential that people see in genetic engineering. They haven’t solved the food problem in general. We have to get past the current generation of crops,” he says.

For Lemaux, the challenges lie not in the details of the science, but in whether new GM crops can actually be brought to market. “It isn’t a problem of us being able to do it. It’s the cost of all of the regulatory hurdles and all the [intellectual property] issues that must be addressed in order to commercialize. This goes way beyond just how you introduce the gene,” she says.

Lemaux has reason to be skeptical–the particular form of rDNA that became standard for transforming plants decades ago, found in a group of bacteria known as Agrobacterium, was patented by Monsanto in August 2013. Lemaux is visibly dismayed. “These are techniques. This is what I have not been in favor of from the beginning. My thought is that we should take all the technologies and put them in the middle of the room. You use them to create a product, and it’s that product that you patent. That’s how the pharmaceutical industry used to be. You didn’t patent all the little parts that go into making an antibiotic, just the antibiotic itself. The situation with GM crops is completely different. Large agrochemical companies, who own most of the patents, hamstring academics, and small companies by controlling the enabling technologies.”

Luckily, basic research carried out here at Berkeley has finally given the field of genetic engineering a tool that allows scientists to hone in on genes already present in an organism, and edit them subtly and precisely: CRISPR/Cas9. Though GM is just a technology, and varies from mutagenesis to rDNA insertions, gene editing might enable the development of a new generation of GM crops. No single technology can quell all of the public’s skepticisms about biotechnology in their food, but scientists at Berkeley are determined to show that there is a future for GM in agriculture.

Design: Leah Anderson; content: Levi Gadye. Image credits: DNA: Ryan Somma; mice: Maggie Bartlett, NHGRI (NIH); insulin: Mr. Hyde/Wikimedia; gene gun: Angela Kaczmarczyk; papaya: Ken (Brave Sir Robin); rice: International Rice Research Institute; CRISPR protein: Jawahar Swaminathan/European Bioinformatics Institute

A GM game-changer?

The dream of many scientists is that, some day, their work will blossom into an idea or technology that will change the world. When Berkeley microbiologists discovered a puzzling genetic system that provided bacteria with immunity against viruses, the lab of Berkeley professor Jennifer Doudna was up for the challenge of solving that puzzle. Many types of bacteria harbored swaths of DNA containing “clustered regularly interspaced short palindromic repeats” (CRISPR). These were broken up by unique spacer regions resembling “fingerprints” of past viral invasions. Coupled with a class of CRISPR-associated (Cas) proteins, these genetic elements allowed bacteria to fend off viral infection. Mystery still remained: just how did Cas proteins, along with CRISPR, so efficiently carry out germ warfare?

While investigating one of three types of CRISPR/Cas immunity, the Doudna lab discovered that a particular Cas protein, Cas9, was responsible for cutting viral DNA in half, destroying the virus. RNA created from CRISPR spacers guided Cas9 like a homing missile to incoming viruses. Better yet, Doudna’s lab learned that they could engineer their own guide RNAs to target any gene of interest, and Cas9 would efficiently cleave that gene.

Scientists had long dreamed of an enzyme that would easily cleave specific genes at precise locations, creating a “double-stranded break” (DSB), because this would allow for site-specific gene editing, a previously labor-, cost-, and time-intensive process. The Doudna lab had struck gold: Cas9 was the holy grail of genetic engineering.

Thus began the CRISPR craze. Within 16 months, dozens of labs worldwide showed that Cas9, along with the guide RNA design invented in the Doudna lab, could be used to mutate individual genes in cell lines, mice, fish, flies, plants, and, most recently, even monkeys.

The process was quite simple: introduce Cas9 along with guide RNA into early-stage embryos, or mammalian or plant cells, and voilà, the gene of interest was mutated. Quite often, the desired DSB would occur in the correct gene, leading to a slight mutation that would be wholly inherited upon growth of the organism or cell culture. With this technology, biologists could introduce disease-causing mutations into human cells or model organisms, screen cell lines en masse by testing out mutations all across the genome, and even selectively modify individual genes in crops. The question still remained whether more precise genetic engineering could kickstart a new generation of GM crops that appealed to the general public.

Design: Helene Moorman; text: Levi Gadye; grapefruit: Aleph; papaya: Mark Peters; corn: Mgmoscatello; cassava: Neil Palmer

GM to Order

Though his lab’s GM tomato remained in limbo, Staskawicz sensed that CRISPR/Cas9 might be the tool for the next iteration of disease-resistant GM crops. CRISPR/Cas9, once it is functional in an early, embryological plant cell, only causes minor changes to pre-existing genes–and the bacterial CRISPR/Cas9 components do not linger after those changes are made. “At the end of the day, we aren’t putting anything extra in[to the genome]. We’re deleting out a few nucleotides. We feel that it’s going to be a safe, precise technology,” says Staskawicz.

Viruses can present a serious challenge for farmers of numerous crops worldwide. Although some scientists had successfully used traditional gene insertions to protect certain crops from viral disease, Staskawicz, along with plant and microbial biology graduate student Michael Gomez, devised a strategy to use CRISPR/Cas9 gene editing to provide simple viral resistance to a crop in dire need, cassava. The cassava plant, constituting the “third most popular source of calories in the world,” according to Staskawicz, was being ravaged by cassava brown streak disease (CBSD), a viral disease, and was thus a prime candidate for GM.

Brian Staskawicz and Michael Gomez examine a cassava plant. Gomez is working to engineer disease resistance into cassava using CRISPR/Cas9 gene editing.

Gomez is engineering a mutation in a cassava gene that will foil the CBSD virus from using cassava proteins for viral replication. First, he is optimizing CRISPR/Cas9 to model CBSD resistance in Arabidopsis thaliana, the model organism of plant biologists. "The next step would be generating transgenic cassava lines that carry the CRISPR/Cas9-induced mutation,” he says. “Something I'd like is to follow through and see these cassava lines field tested in Africa for disease resistance.” He will enjoy support for this project not just from his lab, but also from a $10,000 award recently bestowed by the Dow Sustainability Innovation Student Challenge.

Unlike earlier site-specific gene editing techniques, which depend on designing unique, DNA-cleaving proteins for each gene of interest, CRISPR/Cas9 uses the same protein (Cas9), steered by a cheap, readily-made guide RNA, making this technique “super easy,” Gomez says. When technology makes science easier, discoveries sometimes snowball–and Gomez may very well be riding at the top of a new wave of CRISPR/Cas9 innovations in plant biology and GM agriculture.

Bringing CRISPR to Crops

The GM controversy may remain unresolved for some time, but Berkeley scientists are hopeful their projects will make it out of the lab and into a world in need of agricultural innovation. Lemaux, having already dedicated so much energy into GM food crops that were never given their chance to shine, is shifting gears. “My lab is now involved in bioenergy. We’re engineering tobacco to make what they call ‘drop-in’ fuels, which are fuels extractable from leaves that you can use directly as gas or plane fuel,” she says. GM tobacco destined for fuel would avoid the regulatory mess surrounding GM foods; in fact it has governmental support from the Department of Energy. “It’ll be interesting to see how such engineering in a non-food/non-feed crop for bioenergy plays out in the marketplace,” says Lemaux.

The Staskawicz lab, on the other hand, is betting on CRISPR/Cas9 to invigorate GM crop invention and success, by engineering small but potent changes in pre-existing plant genes. Some of these changes are “so small that they could have occurred naturally,” according to Gomez, potentially increasing their public appeal. For this reason, CRISPR/Cas9-edited crops may avoid classification as true GM crops, which are often defined based on possession of foreign DNA. The EU, long a thorn in GM agriculture’s side, is considering classifying gene-edited crops not as GM, but as the products of New Plant Breeding Techniques, or NPBTs. Other countries, including the US, might very well follow suit. The irony of the arbitrary nature of these designations isn’t lost on Eisen. “They should’ve just called GMOs NPBTs,” he says.

Though regulatory hurdles must be cleared before Gomez’s cassava makes it into the hands of farmers in Africa, Berkeley is home to the inventors of CRISPR/Cas9 gene editing, and therefore the owners of this technology and its related patents. Doudna, along with her co-inventors, founded a biotech company called Caribou Biosciences last year, in order to house the intellectual property of CRISPR/Cas9 at the interface of academia and business. Rachel Haurwitz, co-founder and CEO, hopes Caribou will enable all scientists, from academic labs to biotechnology firms, to readily apply CRISPR/Cas9 to their problem of choice. “The reality is, for most people, the genome engineering part isn’t the exciting part,” she says. “It’s a path to doing very interesting biology, it’s a path to making the modified cells or organisms they need to do their basic or even applied research.” Perhaps it is a path to science reaffirming its positive presence on farms—and towards GM food securing a more welcome place at the dinner table.

Design: Leah Anderson; data source: Fernandez-Cornejo et al. Genetically Engineered Crops in the United States. USDA/ERS Report No.162

Featured image: Professor Brian Staskawicz and Michael Gomez examine a cassava plant. Gomez is working to engineer disease resistance into cassava using CRISPR/Cas9 gene editing.

credit: David Galvez/PMB