Disease-resistant Cavendish bananas, protein-enriched soy flour, and reduced-lignin alfalfa—these are a few of the gene edited plants that are entering field tests. Such plants may succeed in arresting diseases, increasing nutritional value, and reducing agriculture’s environmental footprint.

As the global population rises to unprecedented heights, society needs sustainable, resilient crops more than ever. But traditional breeding practices take time to implement, and genetically modified organism (GMO) varieties cost over $100 million. Gene editing, however, opens new opportunities by accelerating the introduction and reducing the cost of enhanced crops. With gene editing, no foreign DNA is introduced. Instead, relatively subtle changes to DNA are made, changes that are all but indistinguishable from the DNA changes that occur in plants naturally or in mutagenesis.

Attracting consumers, resisting disease

Backed by a $25 million investment from Deerfield and Bayer Leap, Durham-based Pairwise Plants is using CRISPR to make nutritious fruits and vegetables, such as kale and mustard greens, more appealing. Pairwise also has a $100M investment from agricultural giant Bayer focused on staple crops such as corn, soybeans, wheat, canola, and cotton.

The partners are just two of the firms that are exploring ways genome editing can benefit agriculture. For example, another firm, Corteva Agriscience, believes that genome editing can enhance disease resistance in crop plants. According to Neal Gutterson, PhD, Corteva’s senior vice president and CTO, native genes that confer disease resistance can be found among the varieties of a plant species. However, these genes are seldom found in the highly adapted and high-yielding varieties used in production agriculture.

“Genome editing,” says Gutterson, “can move these resistance genes into the genome of the elite production varieties.” Corteva also focuses on consumer-oriented traits, such as improving the nutritional value of grain for food and feed uses. Increasing the availability or quality of the protein meal derived from grains will help increase animal productivity as well as the growing demand for protein while decreasing resource use.

Significantly, the company can now enable edits directly in elite production varieties. In addition, the company is working to expand the toolkit of Cas enzymes with distinct PAM recognition sequences through a partnership with CasZyme. The partners’ hope to open more of the genome to editing, as well as to enable more effective edits.

“We actively advocate for scientifically appropriate, risk-proportionate policies to regulate genome edited products,” Gutterson declares. “With the notable exception of the European Union, many jurisdictions have announced policies that encourage the application of this plant breeding innovation.

“We believe our work internationally has been valuable in supporting regulators as they arrive at scientifically grounded and risk-based policy decisions and treat genome edited crops not containing novel combinations of genetic material similarly to traditionally bred crops.”

Expanding TALENs

Gene editing technology that harnesses transcription activator-like effector nucleases (TALENs) is being applied by Calyxt, which intends to “enhance the unique characteristics that naturally exist in each plant.” The company, which has exclusive access to a proprietary TALEN technology for use in plants, recently licensed a gene editing technology that originated at the University of Minnesota and promises to increase the efficiency with which gene edited plant products are developed.

The method was co-invented by Dan Voytas, PhD, co-founder and chief science officer at Calyxt and professor of genetics, cell biology, and development at the University of Minnesota. It has the potential to reduce editing timelines from approximately one year to several months.

The technology delivers developmental regulators and gene editing reagents to somatic cells of whole plants. This induces meristems that produce shoots with targeted DNA modifications; gene edits are transmitted to the next generation, sidestepping the need for tissue culture.1

“This breakthrough,” asserts Jim Blome, CEO of Calyxt, “will enable editing in some crops of interest that have been traditionally difficult to edit, and adds speed to the process.”

Calyno™, high-oleic soybean oil, is the first and only gene edited food product on the U.S. market. Calyno beans were originally grown and processed by Calyxt, which was also the first to market the oil. The company now intends to sell the beans to processors to do the mechanical crushing, refining, and distribution.

According to Blome, Calyxt is expanding its product line beyond the high-oleic soybean product. The company has five product candidates that it plans to launch in the next few years. For example, a hemp product is scheduled to launch in 2020; an alfalfa product in 2021; and a high-fiber wheat product in 2022.

The alfalfa with reduced lignin content, which was developed in collaboration with S&W Seed, may improve the efficiency of livestock feeding. “You can feed cows less,” Blome explains, “because they digest and absorb more of the available alfalfa, reducing waste and resulting in a lower environmental footprint.”

Hemp, another promising crop, presents economic hurdles that may discourage growers. “We want to eliminate some of the problems that keep hemp from being broadly planted,” says Blome. “For instance, our technologies are being applied to hemp to bring more uniformity to growers’ fields—reducing variability, lowering risk, and
improving yield.”

Harnessing noncoding genes

Bananas are the fourth most important global food crop, whereas coffee is the most consumed beverage after water. These crops provide livelihoods for hundreds of millions of people. But breeding is not a viable alternative. A new coffee variety can take 25–30 years to develop. Store-bought bananas are asexual. In fact, gene editing is the only practical way to make a change in bananas.

Initially, Tropic Biosciences performed standard gene knockouts in the development of products, such as coffee beans with low caffeine content and a banana that produces less ethylene and therefore ripens more slowly. Subsequently, the company supplemented its knockout capability by developing the Gene Editing induced Gene Silencing (GEiGS) platform, which allows a sophisticated use of gene editing for some very attractive applications, including crop protection against viruses, pests, and fungal disease. GEiGS is a combination of gene editing with RNA interference.

Gene Editing induced Gene Silencing
Tropic Biosciences has developed Gene Editing induced Gene Silencing (GEiGS), a platform that is used to redirect the silencing function of existing noncoding genes. By changing only a few nucleotides, GEiGS can fine-tune expression levels and thereby control the tissue in which the silencing occurs, enabling highly customized applications.

“We use GEiGS to edit noncoding genes that control the work of the other genes by making precise and specific changes to only a few nucleotides,” says Gilad Gershon, CEO, Tropic Biosciences. “By changing a very low number of base pairs, we redirect the silencing function of existing noncoding genes.

“If a microRNA today silences gene A, we can make it silence gene B, downregulate an entire gene family, or target a gene within a virus, for instance. Instead of knocking out a gene, we can knock it down—by 10, 20, 50, or 70%—to fine-tune the expression levels to minimize any deleterious effect.”

Tropic Biosciences is using GEiGS to develop banana plants that attack the soil-borne fungus (Fusarium oxysporum) that causes Panama disease, a blight that has been devastating banana production across the globe. Although the fungus is resistant to fungicides, it may be stopped by Tropic Biosciences’ Cavendish banana plant, the company’s flagship product.

“We take pride in using technology to solve big problems and to support the livelihoods of 25 million coffee growers,” adds Gershon. “Coffee growers are some of our planet’s poorest people.”

Exploiting metabolism

Conventional genetic screens are costly and time-consuming. They evaluate thousands of potential targets but produce few “hits.” To improve target identification, Yield10 Bioscience developed GRAIN, a novel gene discovery platform.

“We use metabolism to rationally mine genomic data,” says Kristi Snell, PhD, Yield10’s chief science officer and vice president of research. “Most traits that are worked on involve some type of metabolic change, so pinpointing changes to optimize pathways is very powerful.

“We ask what metabolic changes a plant needs to achieve the desired outcome. Then our sophisticated metabolic models, which encompass all the chemical reactions in a plant, quantify what combination of changes needs to occur. Next, we use the genome-mining module to provide a ranked list of a small number of gene targets from the input conditions.”

Gene Ranking Artificial Intelligence Network (GRAIN)
The Gene Ranking Artificial Intelligence Network (GRAIN) from Yield10 Bioscience is a yield improvement discovery platform that evaluates combinations of plant gene modifications. It analyzes genomic data about metabolic flux to identify which combinations are most promising, that is, which genes should be edited to improve carbon conversion efficiency.

According to Snell, GRAIN can be used to optimize anything with a metabolic underpinning. Yield10 Bioscience has worked extensively with camelina and canola plants.

Camelina has a unique oil configuration with a particular mega fatty acid that makes the oil healthier and that has potential as a feed supplement for salmon and other farmed fish. Yield10 is working on improvements to make the crop’s growth economically feasible in the United States by increasing the native oil and also by using camelina to produce value-added products, such as biopolymers or specialty oils.

The company’s first commercial product is a camelina line that has been edited in three different genes to produce a 4.7% increased seed oil content, an 11.8% increase in oil per individual seeds, and larger seeds. This line is deemed unregulated by the U.S. Department of Agriculture (USDA) and is being scaled up. Three other sets of gene edited camelina lines have gone through the USDA evaluation process, and a fourth recently successfully completed the process.

Maximizing protein

“We plan to pack more nutrients, particularly protein, into plant-based food and feed crops,” declares Lloyd Kunimoto, CEO, Amfora. The company intends to bias plants toward protein production by manipulating NF-YC4, a gene that is highly conserved across many plant species. It acts like a switch. When it is on “high,” it favors the production of proteins; when it is on “low,” the production of carbohydrates.

Amfora can delete specific pieces of DNA from the NF-YC4 sequence that keep the switch set to low. Absent these pieces, the switch remains on high, allowing production of up to 20% more protein than the natural ratio. Plants remain healthy, and yields are unaffected.

“In principle we can create a high-protein version of any crop, and we have demonstrated the technology in corn, soy, alfalfa, and rice,” asserts Kunimoto. “We are excited about our most advanced crop, soy, because of its potential economic and environmental impact. Part of our funding is from the United Soybean Board to improve the crop to benefit all U.S. growers and [increase production in] the 98-plus million U.S. acres [devoted to growing soybeans].”

Soy is the primary protein source in animal feed as well as in the human diet in many parts of the world. As seeds have been built for higher yields, the protein content has drifted down.

Amfora plans to turn the soy into a high-density protein source for plant-based meat—eliminating the need for a capital-intensive concentration process—to produce a more environmentally friendly raw material. Another potential application is aquaculture feed, especially for farmed fish that need a dense protein dietary source. The typical source, fish meal, is not sustainable.

Editing organelles

In plant cells, the energy-producing organelles known as mitochondria and chloroplasts are well protected by their cell membranes—so well protected, in fact, that they are usually impermeable to gene editing agents, such as those found in CRISPR systems. The membranous barriers that organelles pose to gene editing may be overcome by Edit Plasmids, a unique technology developed by Napigen.

Edit Plasmids
Edit Plasmids, a holistic gene transfer technology developed by Napigen, can deliver gene editing components to mitochondria and chloroplasts, organelles that usually resist the import of nucleic acids. By editing mitochondrial genomes, Napigen hopes to produce hybrid, high-yielding crops and even contribute to applications beyond agriculture.

Edit Plasmids uses biolistic gene transfer and plasmids to get the CRISPR gene editing components into the organelles. Proof of concept was demonstrated in yeast mitochondria and in Chlamydomonas chloroplasts.2 In a recent study, Edit Plasmids were constructed with two expression cassettes, one for the expression of Cas9 and the other for the expression of guide RNAs. Cassettes contained a unique set of organelle-specific promoters and donor DNA.

“We are interested in targeting mitochondrial DNA in wheat and rice because certain mitochondrial genes are associated with male sterility,” states Hajime Sakai, PhD, CEO, Napigen. Male sterile plants can accept pollen from other plants and allow the production of hybrid seed. Hybrid seed can produce plants with increased yield, a process known as hybrid vigor. Although hybrid crops are the best way to increase yield, the current techniques can be applied only to a few crops.

“For example, wheat has male and female parts in the same flower, making it difficult to create male sterile plants and hybrid seeds. You need a genetic solution,” notes Sakai. “Mitochondrial engineering has been nonexistent because of the DNA transmission problem and lack of a selection marker. Our technology allows us to investigate how mitochondria can be utilized in plants.”

Mitochondria present another challenge. The DNA multiplies, leading to the accumulation of many copies within each mitochondrion. For a stable change, all copies need to be edited. Similar to the gene drive system used in mosquitoes, Napigen’s Edit Plasmids keep changing the wild-type DNA into the edited version.

“After we published our results, we fielded inquiries from people dealing with rare human mitochondrial diseases, and we received an award from the United Mitochondrial Disease Foundation to investigate human applications,” reports Sakai. “Our ultimate goal is to develop molecular tools to study mitochondrial DNA functions and to contribute to science beyond increasing yields in plants.”

 

References
1. Maher MF, Nasti RA, Vollbrecht M, et al. Plant gene editing through de novo induction of meristems. Nat. Biotechnol. 2019; 38(1):84–89. DOI: 10.1038/s41587-019-0337-2.
2. Yoo BC, Yadav NS, Orozco EM, Sakai H. Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts. PeerJ 2020; 8: e8362. DOI: 10.7717/peerj.8362.

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