GEN BiotechnologyVol. 2, No. 1 News FeaturesFree AccessSalad Days: Pairwise Gene Edits Food to Topple Nutrition BarriersJonathan D. GrinsteinJonathan D. GrinsteinE-mail Address: jgrinstein@liebertpub.comSenior Editor, GEN Media Group.Search for more papers by this authorPublished Online:15 Feb 2023https://doi.org/10.1089/genbio.2023.29078.jdgAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Under the brand “Conscious Foods,” the food tech gene editing company plans to broaden nutritious options by making fruits and vegetables fresher, tastier, and easier to access.Conscious Greens up close. The salad greens come in a range of colors and leaf shapes adding variety to the salad category. Pairwise has developed their Conscious Greens salad mixes using CRISPR. (Credit: Pairwise)Food scientist and entrepreneur Haven Baker's children are sick of kale. “My kids don't want us to buy more salad kits with kale—they don't want to eat it!” Baker told GEN Biotechnology. “They want to eat romaine lettuce.”Baker's kids are not alone. In households across the United States, kids and adults alike want nothing to do with the leafy superfood. “While things like kale have a lot of nutrition, most people don't want to eat [bitter] greens,” said Baker, the cofounder and chief business officer at the food tech company Pairwise. “People want to eat lettuce, especially iceberg and romaine, which pack almost no nutrition.”A little more than half of the population in the United States eats their recommended daily allowance of vegetables, whereas 1 in 10 people simply do not have access to such luxuries. Despite all the best efforts in public education and other focus areas, these trends have not improved much for decades. Baker and the team at Pairwise aim to build a healthier world by using CRISPR genome editing to develop novel nourishing foods that lower barriers to fruit and vegetable consumption, such as taste and accessibility. “We're working on improving convenience and nutrition, increasing snacking ability, and making consumption easier,” Baker said.Haven Baker, Cofounder and Chief Business Officer at PairwisePairwise believes the use of technology offers a transformative opportunity to open a new horizon for the $66 billion U.S. retail produce market and is currently developing new fruits and vegetables. In 2023, Pairwise hopes to follow in the footsteps of Sanatech's genetically edited Sicilian Rouge tomatoes being released into the Japanese market in September 2021 (Box 1). Rooted in North Carolina, Pairwise will sell several produce varieties “improved” by gene editing under the brand name Conscious Foods. The first product is Conscious Greens, which will be salad blends (Fig. 1). Heading to the market after that are seedless blackberries and pitless cherries.FIG. 1. Conscious Greens brings new offerings of greens with superior nutrition to current salad options. (Credit: Pairwise)Pairwise announced $25 million in Series A funding in March 2018, led by Deerfield and what is now Leaps by Bayer, and $90 million in Series B in February 2021 to develop its gene editing platform and initial product portfolio. In 2018, the company also announced a $100 million collaboration with what is now Bayer Crop Science to advance gene editing tools in corn, soybeans, wheat, canola, and cotton.Pairwise was launched in 2017 by a familiar trio of CRISPR gene editing experts: Broad Institute colleagues Feng Zhang, David Liu, and Keith Joung, an investigator at Mass General Hospital. The three friends were also cofounders of Editas Medicine, the first CRISPR biotech company to go public. Although these principal investigators are not exactly known for their prowess with plants, the utility of CRISPR and the untapped potential of genome editing in agbiotech made it almost inevitable that they would apply their expertise to farming rather than pharma.To zero in on potential commercial products, Pairwise looks at the intersection of three perspectives: consumer benefit, scientific feasibility, and return on investment (ROI). The initial step is to find patterns in consumer data for what people want to eat and why. They have found that not all fruits and vegetables fit people's modern lifestyles. For example, the introduction of baby carrots in the 1980s led to an increase in carrot consumption because they were easier to snack on and add to salads.Box 1. From Garden to Sea: Japanese Approved CRISPR-Edited ProductsSanatech Seed Co, Ltd., a start-up company partnering with the University of Tsukuba, has been selling gene-edited tomatoes directly to consumers in Japan since September 2021. These tomatoes have been genetically altered with CRISPR to contain high levels of γ-aminobutyric acid (GABA). The Tokyo-based manufacturer received a parental line of the Sicilian Rouge variety from Pioneer EcoScience and developed the variety “Sicilian Rouge High GABA” with enhanced GABA content through gene editing. Taking GABA orally, according to Sanatech, can lower blood pressure and promote relaxation.In addition, CRISPR-edited food has made the jump from the produce aisle to the seafood counter in Japan. In October, the country approved two CRISPR-edited fish: a red sea bream with more muscle growth and a gene-edited tiger puffer with less hunger. The fish were developed through a collaboration of the Kyoto-based Regional Fish Institute, Kyoto University, and Kinki University, and was overseen by the Japan Ministry of Health, Labor, and Welfare and the Ministry of Agriculture, Forestry, and Fisheries.Both fish grow bigger than their wild counterparts. The genome-edited tiger pufferfish grows faster and gains 90% more weight in the same amount of time as the traditional puffer. This allows a shortening of the farming period from the conventional 2 years. Sliced pufferfish sets are sold through the website in various sizes and prices.The genome-edited red sea bream has 20–60% more edible yield, whereas feed utilization efficiency was increased by ∼14%. It was introduced to the press under the nickname “Muscle Madai” (madai means red sea bream in Japanese) but is being sold by Regional Fish Online as “22nd Century Sea Bream.” A set of ∼16 thin slices on a bed of dried kelp is sold in Japan for ¥3,000 (or about $22).The Regional Fish Institute does not plan to stop there and announced—along with Kyoto University and Tokyo Marine University—being awarded a government subsidy of ¥97.5 million (over $710,000) for research as a “year-2022 growth-type small and medium enterprise R&D support project.” The company will use the money to develop gene-edited tilapia and vannamei shrimp.Box 2. The Tail-End of Transcription Activator-Like Effector Nuclease Food Products?Preceding DNA editing methods, like transcription activator-like effector nucleases (TALENs), have been and may still be used to make food ingredients available to consumers. Both CRISPR and TALEN are highly specific, producing few off-target editing (cleavage) effects (thus preventing undesired cell mutations), and can be fused to other functional protein modules for versatility in gene manipulation. Both have also been reported to be superior in specific applications, but CRISPR's popularity has grown among researchers because the smaller size of the tailored part makes them simpler to design and cheaper to use.There have been a wide range of TALEN users from large commercial seed companies (e.g., Bayer, Syngenta) to small biotech companies (e.g., Simplot, Epicrop) to enhance the efficiency and precision of the crop improvement process. A few TALEN-edited crops have made it to market, such as a TALEN-edited soybean oil without trans fats—high oleic low linolenic (HOLL)—was released to the market by Calyxt, a plant-based technology company, in 2019. In June 2020, Calyxt announced that its HOLL soybean was deemed a nonregulated article by the Biotechnology Regulatory Services of the Animal and Plant Health Inspection Service (APHIS), an agency of the United States Department of Agriculture.Similarly, in April 2021, APHIS is extending deregulation to J.R. Simplot Company's (Simplot) potato variety developed using genetic engineering, designated as Snowden Z6 (Z6 potato). The potato variety is engineered for late blight protection, lowered reducing sugars, low acrylamide potential, and reduced black spot bruising. After considering the public comments, APHIS has determined Z6 potato is unlikely to post a plant pest risk and is extending deregulation to the Z6 potato line.Just as with CRISPR, TALEN has been applied not only to produce but also to animal products. A 2019 study in Japan, “Targeted mutagenesis of the ryanodine receptor by Platinum TALENs causes slow swimming behavior in Pacific bluefin tuna (Thunnus orientalis),” the Platinum TALENs method of gene-editing was used rather than CRISPR to induce mutations in Pacific bluefin tuna. The change reduced the “burst swimming” ability of the tuna.As a result, when the bluefin were disturbed by touching them, they swam away slowly, rather than with a burst of speed. Farm-raised tuna with burst swimming edited out may be less likely to break their necks by swimming at high speed into the walls of tanks and net pens, a common cause of mortality. The trait has not yet been commercialized, but Japan's farmed bluefin sector gained stronger footing in 2021, giving those pursuing the technology a strong incentive to push their progress.Baker says that the data show that consumers want freshness and consistency, yet the quality of many fruits and vegetables changes from week to week or season to season. “If you look at blueberry consumption, it has gone up three to four times in the past 25 years, largely due to being made available year-round,” Baker said. “Are there any summer fruits that we would eat more of if they were available in good quality year-round? The response is, absolutely!”Crisper with CRISPRThere is a lot of traditional breeding that has been employed in increasing production or making life easier for farmers, but more effort needs to be focused on making better food products for consumers faster.“If you're a cherry farmer, you're worried about disease and yield, but if you're a consumer of cherries, you'd really like to get rid of the pit and have them available year-round,” said Baker. “Sometimes, these products could be created through conventional breeding, but most of the time, if it could have been done within 50 or 100 years, it would've already been done.”With the transformative potential of CRISPR, Pairwise is creating new crops based on consumer research. “We take these potentially viable products and use CRISPR to accelerate and bring those traits forward. With CRISPR, we can approach some of these fundamental problems that have hampered consumption and availability.”Pairwise has also put a lot of money into developing technology that can quickly come up with a product idea for consumers. The company has raised $115 million in two funding rounds to help it improve gene editing technology for row crops and other plant-based uses. “We are innovating the editing technology to make new products or more products than we could two years ago,” said Aaron Hummel, vice president of research and development at Pairwise.Genetically Modified Organism IntoleranceThe Pairwise team is alert to the fact that previous attempts at plant genetic engineering have not been successful (Box 2). Consumer acceptance of their products' use of CRISPR technology appears to be aided by the fact that these crops are not classified as “genetically modified organisms” (GMOs). Rather, they are referred to as “genome-edited.” To this end, there is no mention of GMOs, which typically refer to the practice of transferring genes between organisms and worry many consumers, on the Pairwise and Conscious Food websites.This change in terminology may have significantly lessened the historical backlash against bioengineered plants. Recent studies show that attitudes toward gene editing in agriculture are changing (at least in the United States), but most of the people who took part in the studies said they knew very little or nothing about the practice. People have heard of and are enthusiastic about CRISPR. When asked without more information, about half of the people who took part in the study agreed that gene editing is good for agriculture. However, after reading a short introduction, nearly two-thirds of them felt the same way.According to Sarah Davidson Evanega, lead of stakeholder communication at Pairwise, the biggest confusion really revolves around language. She claimed that, despite their similarities in sound, genetic engineering and gene editing are very different in terms of how they are carried out. It serves as a reminder of how crucial using the appropriate language is.Aaron Hummel, Vice President of Research and Development at PairwiseMany of Pairwise's products made with CRISPR require investigation to untangle the genes and pathways behind plant traits. Hummel said that plant tissues and biochemical pathways are very different in their complexity. Some of Pairwise's plant variants are very simple, controlled by a single gene, and require just one edit to make them. But in other cases, the trait in question is multigenic, which means that several genes have effects that add up or work together. In these cases, it takes more than one edit to get the right phenotype.Sometimes, plant biology is not fully understood and requires more discovery work. Hummel says that there have been cases wherein Pairwise has created a series of edits at a specific target gene, followed by phenotyping to see which variants best expressed the sought-after trait. Or sometimes, they might be in the dark about which gene to target. In these situations, Pairwise researchers usually assemble a set of gene targets to alter and test how they affect the target tissue. Either way, when doing this kind of discovery work, Hummel says they typically measure the trait in question and a couple of effects where the plant might compensate negatively.The Not-So-Mean Mr. MustardPairwise's Conscious Greens are an example of how they do research and development to make new and better fruits and vegetables. With this product, the innovation is making mustard greens that are healthy, do not taste spicy, and do not have a strong smell. Pairwise found that consumers want more nutritious options for greens because they do not like the taste of bitter greens. Pairwise was looking for ways to make salads healthier than just lettuce, so they focused on mustard greens, which have very healthy leaves but are too pungent for most salad lovers. The potential ROI also looked promising: packaged salads are a $10 billion industry and one of the largest parts of the supermarket produce aisle.Chewing mustard greens produces compounds known as allyl-isothiocyanates, which are pungent and antiherbivory (i.e., defense mechanisms against herbivore attack). As cells rupture from chewing, two physically separated molecules—a myrosinase enzyme and a glucosinolate precursor—mix and create allyl-isothiocyanate, also found in other pungent plants such as horseradish. But when cooked, the myrosinase enzyme gets broken down (denatured), preventing the reaction in the mustard greens' leaves.Hummel says that the team at Pairwise used gene editing to knock out the myrosinase gene, mimicking the effect of cooking and preventing the reaction that creates bitterness from occurring. Removing the gene to eliminate pungency required a fairly simple edit: a frameshift in the coding sequence, resulting in a loss of protein function.But it was not all straightforward. “The complex part of developing this product is because the mustard species is tetraploid, which led to many more targets in the genome that we had to knock out all simultaneously to get the full non-pungent effect,” Hummel said. “The edit was straightforward, but getting the right phenotype was pretty complex. The result is that the leaves are now nonpungent, and there's a fairly pleasant flavor—it tastes relatively similar to lettuce, and the texture of the leaf is a lot like lettuce. So, we're very excited about this as a better salad product.”Miniaturized Mangoes and MoreBaker says Conscious Greens will be available in select grocery stores and restaurants in the United States in 2023. “We plan on launching Conscious Greens in regional retail markets next summer. We are now finishing product development and doing tests where we grow these in fields and run them through processing plants.”Pairwise has sent out thousands of free samples to get feedback from customers, in part to find out if they have got the right crunch and color. Conscious Greens will have more than one edited variety of mustard greens, all of which should help make a very healthy salad mix. Patrick Hsu, cofounder of the Arc Institute and assistant professor of bioengineering and Deb Faculty Fellow in the College of Engineering at the University of California, Berkeley, tweeted last summer that he was excited to try the world's first CRISPR-edited salad from Conscious Foods.In a decade or so, Baker envisions Pairwise selling bite-sized, healthy, and flavorful fruits and vegetables—mangos the size of cherries with edible skins available all year—to replace chips and Snickers as the snacks of the future. And he hopes that products like this will be joined in the produce section by many other foods and vegetables made by other CRISPR produce companies.“This is going to change, hopefully in a positive way, all fruits and vegetables and eventually agriculture in a positive dimension. It will enable adaptations to climate change, disease resistance, and better profits for growers. There's a growing recognition that we will need [gene editing] technology to solve these problems, especially in an era of food inflation. We need more productivity.”FiguresReferencesRelatedDetails Volume 2Issue 1Feb 2023 InformationCopyright 2023, Mary Ann Liebert, Inc., publishersTo cite this article:Jonathan D. Grinstein.Salad Days: Pairwise Gene Edits Food to Topple Nutrition Barriers.GEN Biotechnology.Feb 2023.5-9.http://doi.org/10.1089/genbio.2023.29078.jdgPublished in Volume: 2 Issue 1: February 15, 2023PDF download
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