CRISPR & Co.: Brave new food

Last year, an exceptional dinner for actors, politicians, journalists and other city celebrities was planned at a haute cuisine restaurant in New York. It was billed as a world first, a meal of the future – the launch of an expedition into an age of new food. On the menu: tofu and soy burger, lightly smoked mashed potatoes, and soy milk and strawberry fontaine­bleau for dessert. Not all that exceptional on the surface…but all were made with ingredients from gene-edited plants designed by biotech company Calyxt. André Choulika, head and founder of the Cellectis Group, Calyxt’s mother organisation, explains that the event mirrored a similar dinner given in Paris 250 years ago: “When we were having chips and fries at

Calyxt made from one of the first harvests of our genome-edited potatoes, I thought: ‘This is a historic event.’” The chips were made from potatoes with a gene that had been edited to inactivate the enzyme responsible for the degradation of sugars in the tuber, keeping them sweeter in cold storage and reducing the formation of the carcinogen acrylamide during the frying process. “While I was thinking that billions of people could eat such food in the future, I was reminded of Antoine Augustin Parmentier,” Choulika says. The 18th-century French botanist shot to fame for his role in making the potato a pillar of France’s food supply. To overcome myths that the foreign tuber was toxic – some skeptics even claimed it transmitted leprosy – Parmentier invited members of the elite class like King Louis XVI and American ambassador Benjamin Franklin to enjoy a range of potato recipes. The sly promoter also had potato fields guarded during the day, but left them unguarded at night, giving farmers the chance to steal the ‘valuable’ plant. It’s hard to say how much those tricks contributed to making potatoes a staple food for hundreds of millions of people in the EU alone. But Choulika decided to revive the idea “to enable a new food revolution that will influence the 21st and further centuries.”

Calyxt’s dinner will probably not convince the European public to put aside its concerns on genetically modified organisms (GMOs) or their gene-edited organism cousins (GEOs), but the event did raise public awareness of new genome editing technologies, and how much they could change humanity’s food supply in the very near future. Practically every major seed company and leading breeder is already pursuing genome editing, at least for research purposes. And a big reason why is the game-changing gene-scissor technology CRISPR/Cas9.

CRISPR – a booster for plant breeding

Take Bayer, for example. If the German giant is given the go to acquire Monsanto, it will control about 40% of the global seed and pesticide market. CRISPR/Cas9 is “a relatively new technology in our plant breeding activities,” says Adrian Percy, Head of Research and Development at Bayer’s Crop Science division.  Even so, the company “has been working on genome editing for a few years now, using different types of nucleases like meganucleases and TALENs” (Transcription Activator-Like Effector Nucleases).
According to Percy, the goal is to speed up development in plant breeding and bio­technology, particularly when it comes to introducing disease resistance or insect resistance traits into new varieties. Another area of high interest is increasing plant resistance to a­biotic stress factors like salinity and drought. “And then, of course, also to more generally help increase plant yield,” Percy adds. In early research projects, Bayer is evaluating the technology, prdominantly for use in cereals, cotton, canola or oilseed rape in Europe, as well as soybeans and also vegetable seeds. “The potential of this technology is just enormous,” Percy says. “I can see this being very, very broadly used.”

CRISPRed corn – made to be waxy

Bayer’s bioengineers want to dramatically shorten development cycles for new varieties, which can currently take up to 10 years or more. With genome editing, Percy hopes to cut that time in half. “To save years on development of new plant varieties is obviously an advantage to us as a business, but also a big advantage to our customers. That’s because we see the emergence of new plant diseases and the impact of volatile weather patterns happening very quickly, and there’s an urgent need to adapt plants to changing climate cycles.”

While Bayer’s genome editing attempts are still in the research phase, DuPont Pioneer has taken things a step farther. The seed specialist has already received USDA approval for a CRISPR/Cas-edited ‘waxy’ corn variety that contains about 97% amylopectin starch instead of the standard 75%. Through the involvement of its subsidiary Danisco, a company that was active in basic research into CRISPR/Cas-systems in nature, DuPont Pioneer holds a couple of CRISPR/Cas patents. It integrated genome editing technology into its breeding programmes over two years ago. “We certainly see this as a technology that we would like to apply essentially in all of our meaningful crops – soybean, corn, oil-seed rape – for Europe sunflower, wheat, rice – you name it,” says Neal Gutterson. DuPont’s vice president of R&D says his firm is focussing on many of the same traits that interest other breeders: “Drought tolerance and better water use are really high-valued traits for our customers,” he reports. “And disease resistance is a trait category that we think is relevant to all kinds of crops and ways to impact yield.” DuPont has also kicked off programmes to improve the value of certain crops, trying to improve for example the quality of oil protein farmers get from certain varieties of soybean.

A SWEET gene to resist blight

Although the companies don’t provide any details about the genes they aim to target with genome editing, there are obvious candidates. Drought tolerance, for instance, could be achieved by increasing the expression of the ARGOS8 gene, which down-regulates the hormone ethylene, an inhibitor of plant growth. Other adaptations to climate change could involve changing when a crop flowers via a deletion of the SP5G gene, which delays the phase. In terms of disease resistance, disrupting the promoter of SWEET genes in rice induces resistance to bacterial blight, and mildew resistance in wheat could be achieved by knock-outs of the MLO-A1 genes. Future targets for increasing yield could be genes involved in the efficiency of photo­synthesis or seed growth and number.

“One of the advantages of CRISPR is multiplexing”

Germany-based BASF AG also recognises the potential of genome editing, but is more reluctant at the moment to publicise details about its development plans. “CRISPR/Cas is a technology that will find broad application in a variety of fields in the life and biosciences,” says BASF Senior VP Plant Science Research Burkhard Kröger. “Applications are in the optimisation of plant traits as well as in industrial biotechnology.” Kröger believes the most important advantage of CRISPR/Cas is the precision of the genetic modification compared to conventional methods like radiation or chemical based mutagenesis. “Genome editing does not induce unwanted side effects in the genome that subsequently have to be bred out via cumbersome, time-consuming crossbreeding.”

Headquartered in Einbeck, Germany,  the KWS Group is already evaluating CRISPR/Cas9 and other genome editing technologies in their research programmes, especially in areas such as improving disease resistance of crops like sugar beet. “One of the advantages of CRISPR is the option of editing more than one target gene,” says Anja Matzk, the firm’s Head of Regulatory Affairs. Such “multiplexing” allows rapid trait-stacking and editing of whole gene networks while the genetic background – the result of hundreds of years of breeding – remains untouched.

Game-changer in  breeding history

This is why genome editing, for the first time in breeding history, could substantially improve complex quantitative traits like drought tolerance or yield. Multiplexing is particularly useful in crops with more than the usual diploid set of chromosomes – among them wheat and corn, which are polyploid. Genome editing allows breeders to target all of the alleles at once, which was hard to achieve with either breeding or classical genetic modification technologies. “CRISPR can be used to change all the alleles in tetra- or hexaploid genomes, which is otherwise very laborious and time-consuming due to the necessity of multiple generations of backcrosses,” Matzk says. Exactly how much time the breeder could save with genome editing, however, depends heavily on the crop. “In terms of breeding, sugar beet is a laborious plant, because it is biennial. With corn, on the other hand, you can harvest three generations in a year.” KWS believes the time savings using CRISPR/Cas would be huge. Instead of six or seven generations, only two or three could be necessary to develop a new variety, Matzk says. “Potentially we could halve the time it takes to progress a breed, which is a substantial improvement.”

Many companies though are still on the fence when it comes to deciding whether Crispr/Cas is the right genome editing system to reach their goals. “Clearly, CRISPR/Cas9 or CRISPR/cpf1 have the capacity to be much more versatile and efficient in their use, so I’m sure that we would gravitate towards these newer technologies,” Bayer’s Adrian Percy says. “But we also anticipate that there will be new versions coming out, because it’s a very fast-moving area of science.” The crop science specialist makes it clear that Bayer will definitely not focus on just one technology yet. “We keep an open mind to all of these technology areas, so that we can pick the right tool for the right application. In some areas, we look more to CRISPR-based systems. With others, where we have more experience, we work with TALENs.” Bayer has licensing agreements with the patent consortium around CRISPR/Cas9-inventors Emmanuelle Charpentier and Jennifer Doudna. BASF decided to reach a global licensing agreement with the Broad Institute of MIT and Harvard for the use of CRISPR/Cas9 genome-editing technology instead – despite the ongoing patent battle between the two groups. “If the patent situation should change, we will react proportionately,” Kröger says, adding that although CRISPR/Cas has “the greatest potential”, the company also keeps an eye on other genome editing methods.

Finger? Crispr? Maybe Talen?

Despite the hype surrounding CRISPR/Cas9, breeders are well-advised not to abandon other genome editing technologies too quickly. For instance, oligo­nucleotide directed mutagenesis (ODM), is one of the simpler methods that doesn’t employ enzymes. It’s been used by companies like Californian Cibus to develop herbicide-tolerant canola, flax, and rice, as well as a Phytophora­-resistant potato. Three European countries – Sweden, Germany and the UK – have categorised ODM as mutagenesis, which is not subject to European GMO regulations. Never­theless, due to a pending decision from the European Commission, Cibus’ varieties are not yet marketed in the EU. Just like plants developed with enzyme-based genome editing technologies like CRISPR/Cas, zinc fingers, and TALENs, they’re in legal limbo. Developed in 2012, CRISPR/Cas is the youngest, cheapest and fastest of these methods, but it may not be the best for editing plant products. “People got excited about CRISPR/Cas because it is very easily designed. Every scientist can design an oligo­nucleotide – the guide-RNA – and can become a gene editor,“ says André Choulika from Cellectis. “We’re gene­-editing geeks who have worked with every new gene­-editing technology that came out in the last 17 years. And we’ve been very excited about CRISPR/Cas, too.“

Thousands of unintended mutations

Originally, Cellectis focused on another, more cumbersome enzyme-based genome editing tool called Meganuclease, then switched to TALEN technology in 2011. Now Choulika has decided not to adopt CRISPR/Cas, due to what he calls “a lack of specificity and efficiency.” What put him off CRISPR/Cas most, he says, were the off-target effects. “CRISPR/Cas is absolutely great to do research, but it is not meant to develop products. We tried it; we don’t like it.“

Target site specificity of CRISPR/Cas genome editing does indeed remain controversial, with evaluation of the frequency of unintended off-target editing in crops showing inconclusive results, at least according to Armin Scheben and David Edwards from the University of Western Australia in Perth in a 2017 Science perspective (DOI 10.1126/science.aal4680). In a May 2017 Nature Methods paper (DOI 10.1038/nmeth.4293), scientists described thousands of unintended off­target-mutations in gene-edited mice after whole-genome sequencing of their DNA. In plants, Scheben and Edwards add, the number of potential off-target edits is even higher than in animals, because many crops are polyploid and have high levels of repetitive DNA. Choulika isn’t convinced by the Australian researchers’ notion that off-target editing can be reduced by using variants of the Cas enzyme and other improvements of the technology. If CRISPR/Cas is so advantagous, he asks, then where is the expected “tsunami“ of CRISPR/Cas-edited plants? “Most recent USDA filings for new varieties have come from Calyxt, and we are a 25-person company…not comparable to Dupont or Bayer,“ Choulika says. “It’s not the fact that we’re better. We just use the better technology – TALENs.“

Instead of focusing on the traits affecting yield, disease and abiotic stress resistances that bigger breeders are fixated on, Calyxt is pursuing a slightly different path. “Most of the effort in biotech­nology tries to focus on the farmer’s needs, in a kind of race for productivity,“ Choulika says. “But we don’t need more yield. We already produce more than we can even consume.“ Hunger, the scientist says, has more to do with wars than plant yield. Calyxt is therefore trying to improve the quality of food, along with the health of both consumers and farmers. Its port­folio includes reduced trans-fat soybean oil, an improved-quality potato, canola oil lower in saturated fat, and low-gluten wheat. And regarding farmer health, it developed  a powdery mildew-resistant wheat – “a pathogen that you usually spray with chemicals,“ Coulika says.

European regulators still haven’t decided whether any of these varieties might one day reach the European market. Lawmakers have delayed a decision for almost two years now. “I’m deeply convinced that they would take a decision to regulate such products reasonably,“ Choulika believes, adding that genome editing is no different than the chemical or radiation mutagenesis that has been standard practice for the past 150 years. “Why should we regulate it differently than every­thing that is already on the market? Everything we eat has been mutated by chemicals or physical means.“ If the EU authorities make the decision to regulate gene­-edited organisms as they have in the past with GMOs that carry cross­species traits, they would “throw Europe back into the Dark Ages, and it will be totally out of the game.“