Introduction – Background and History of Development of Genome Edited Crops –
Since 1996 when “genome editing technology” using zinc finger nuclease (ZFN) was reported, we have been able to take advantage of practical techniques that can “target and modify” genome in living organisms. Then, with the introduction of the “CRISPR/Cas9 system” (Jinek et al. 2012) reported by Doudna and Charpentier in 2012, genome editing has been bringing innovation into various fields including medicine, agriculture, and industry. Especially in agriculture, this technology is attracting attentions and expectations as an approach to modify genes in crop plants efficiently and improve the plants, and to create new “varieties” quickly to help solve various issues and satisfy needs.
Genome editing is not the first technique that humans utilize to modify genes and genomes in crops. We, humans, have been “selecting” and using individuals with beneficial properties for us, such as high-yielding, strength against pests or aridity, and good tastes, since we started cultivation of crops and domestication of animals. In general, such useful traits would be produced by using spontaneous mutations of genes that plants originally have, followed by breeding to create a new mixed generation. That means that human beings have been developing agriculture and building up productive lives and civilization by “selecting” useful traits for agriculture from organisms whose genes had been “mutated”, and utilizing them.
In order to produce lines and varieties with useful mutations for agriculture, however, the traditional breeding has been requiring a long period of time ranging from years to decades, large fields to evaluate many individuals and lines, and labor and costs to manage them. Many techniques, such as DNA marker-assisted breeding and genomic selection, have been developed intending to shorten cultivation time and save labor so far. But when no individuals or lines with traits that meet the purposes (genetic resources) were found, it was necessary to create one with desired mutations using artificial mutation.
Since 1920s when mutagens including radiation and chemical substances were found, breeding using “artificial mutation” which induces genetic mutation artificially was promoted. In Japan, the “Institute of Radiation Breeding” was established in 1960, where gamma ray irradiation was utilized to induce mutation for breeding. Further, ion beam irradiation was applied for breeding at RIKEN. But because mutation occurs randomly in general, there were some hurdles: a lot of labor and cost and a long period of time were required to find out an individual with the desired mutation, and it is hard to induce the targeted mutation in hyperploid crop plants.
Genome editing is characterized by the efficient introduction of desired mutation as it can target and alter a specific site in the genome. For agricultural use, its specific advantages are mainly as follows:
- It can create new targeted mutations in a substantially shorter period of time and with less labor and cost compared with traditional means that look for them from genetic resources or create them with artificial mutation. For this, it has a greater advantage especially in fruit trees which inherently need a long time to breed whose one generation is long.
- By introducing mutations from cultivars with excellent traits, a new variety can be completed in a shorter period of time. When the same mutation is desired to be introduced into many or new varieties, the intended cultivars can be obtained more quickly than with the traditional methods.
- Breeds with useful traits can be obtained by reproducing the mutation revealed in the genetic information of other organisms having the same traits (for example, resistance to pre-harvest sprouting of wheat and red sea bream’s increased muscle mass, which will be described in the next chapter).
- Even when plural genes are targeted, it is possible to mutate them concurrently if they have the same sequences. This would demonstrate a great advantage in genetic modification of hyperploid crops, such as potatoes (mostly tetraploid), wheat (hexaploid), and chrysanthemum (hexaploid). It is quite unlikely to obtain breeds with all of the plural target genes modified at once using natural or artificial mutation, which alters genes irrespective of their sequences in general.
As such, in a wide framework of the breeding system, genome editing is considered basically as one of those techniques to create mutations or materials for breeding. That means that, at least at present, introduction of genome editing would not change the significance of steps including mating and selection in breeding, and it alone would not replace everything in the breeding system completely.
On the contrary, this technology has following drawbacks, which research and development is proceeding to overcome:
- It cannot modify genome unless sequences or functions of the target gene are known.
- Each target crop requires different techniques to introduce genes (DNA), proteins or ribonucleoproteins (protein-RNA complexes) into viable and reproducible cells. Such modification might still be difficult for some crops and cultivars.
- When foreign DNAs (such as Cas9 genes or guide RNAs) are needed to be introduced in the development process of a genome edited crop, the incorporated foreign DNAs must be removed to use this technology as an exemption from regulations for genetic modification. In many crops, it is relatively easy to eliminate the DNAs by breeding, while vegetative reproducing crop plants that can propagate by tree cutting or herbaceous cuttings or with corm have difficulties to have them removed partly because sterility makes interbreeding difficult or properties of the original cultivar change after crossing. “Off-target editing” (introducing mutation into genes or sequences other than targeted positions) is often featured as one of the problems of genome editing, which can surely become a huge difficulty at medical settings if applied. In case of agricultural use (for breeding), however, cultivars are grown from individuals or lines harboring many unidentified mutations. They undergo trait assessments and selections by cultivation tests, and are partly provided as food. As breeding by genome editing also requires trait assessments and selections basically, individuals and lines having problems (such as poor growth, vulnerability to diseases and pests, and bad taste) would be excluded at these stages. Therefore, even if any off-target editing were occurred in a new variety, it would unlikely cause any more problems compared to cultivars developed by the traditional breeding approaches.
Development of Genome Edited Agricultural, Forestry and Fishery Products in Japan
As genome editing increasingly attracting global attention, the government and researchers held discussions on genome editing (as a part of new plant breeding techniques [NBT or NPBT]) in Japan. The Ministry of Agriculture, Forestry and Fisheries set up “the New Plant Breeding Technique Study Group” from 2013 to 2015, and published a report (the New Plant Breeding Technique Study Group 2015) based on the discussions held then. The Science Council of Japan also published a report “Current status and future of genome editing technologies for breeding of agricultural products” (Science Council of Japan, 2014). Parallel to these discussions, the first phase of the Cross-ministerial Strategic Innovation Promotion Program (SIP) led by the Cabinet Office adopted research promotion of genome editing, by integrating studies of genome editing technology, ranging from fundamental technological development to strategies for resource utilization, crop development, and social implementation of mutations. Up to now, some of the results of these studies are leading the research and development of genome edited crops that we describe here. Other developmental studies using research funds including the Grant-in-Aid for Scientific Research (KAKENHI) have been proceeding.
Developmental statuses of specific crop plants are outlined as follows.
Tomato is considered as a representative example in the development of genome-edited crops for practical use in Japan. Particularly, the research group led by Professor Ezura of the University of Tsukuba is leading the development of genome edited tomatoes. Ezura’s group is developing some new traits based on the accumulation of their previous research on mutation. Among them, a plant whose developmental research is most advanced for practical use is the tomato highly containing a health functional component, GABA (gamma-amino butyric acid) (Nonaka et al.2017).
GABA is a functional component known to lower blood pressure and reduce stress. The C-terminal sequence of GABA biosynthetic enzyme covers its own active center normally, while it deforms and uncover the center under stress, leading to activation of the enzyme. By removing the cover via genome editing, GABA was built up by 15-fold in the experimental line (Micro-Tom) (Nonaka et al.2017) and by 3–4-fold in the practical line (F1 from the experimental line and Aichi First) (Lee et al.2018) compared to the wiled type. Tomatoes are commonly known to contain a relatively large amount of GABA. This genome-edited GABA-rich tomato is expected to suppress blood pressure elevation naturally through usual diet. While GABA is a type of amino acids synthesized from glutamine, analysis of the GABA-rich tomato did not show any significant change in the content of other amino acids (Lee et al.2018).
GABA-rich tomato developed by the University of Tsukuba have been drawing attention for its ongoing effort for commercialization. The venture company “Sanatech Seed” launched by the University of Tsukuba is preparing for production and distribution of this tomato.
Besides the GABA-rich tomato, the University of Tsukuba is developing tomatoes with a long shelf life, parthenocarpy (a property of plants including tomatoes and eggplants to bear fruit without pollination) (Shimatani et al.2017), and high sugar content using genome editing techniques. Further, Tokushima University (Ueta et al.2017), Nagoya University, and National Agriculture and Food Research Organization (NARO) (Ito et al.2015) are developing tomatoes with parthenocarpy, high sugar content, and a long shelf life respectively using genome editing technology.
(2) Potatoes (Solanum tuberosum)
Research and development of genome edited potatoes is being conducted by a research group led by Osaka University and RIKEN. The representative target trait of the potato is “reduced content of natural toxin”.
Sprouts and green peel of potatoes contain natural toxins called steroidal glycoalkaloids (SGAs) including solanine and chaconine. Storage management that prevents from increasing SGAs is required during processing and distribution, and it has been a challenge to produce cultivars containing reduced SGAs by breeding. In 2014, Umemoto, a senior research scientist of RIKEN, and his colleagues found that SAGs are biosynthesized from cholesterol, and identified SSR2 as a target gene in the enzyme SSR2 that is associated with biosynthesis of cholesterol (Sawai et al.2014). Further, the research group performed genome editing using TALEN to target SSR2, and revealed that the levels of SGAs were significantly reduced when the potato lost all the functions of four alleles. The finding of this basic study that elucidated a biosynthesis pathway of a secondary metabolite was a breakthrough in the development of crops, which is a typical example showing that elucidation of genetic functions is essential for development of genome edited crops. While the efficiency of introduction of artificial mutation to all of the four alleles might be quite low, genome editing would make it possible to target all of them together if they share common sequences. This study has also demonstrated the availability of genome editing in the breeding of hyperploid crop plants (potatoes are tetraploid).
Many cultivars of potato have difficulty in inbred because of cytoplasmic male sterility. Even if they do inbreed, problems will arise such as presentation of inbreeding depression or alteration of properties of the original varieties because of heterogeneity of the original genetic constitution. As such, it is practically challenging to reproduce good traits of the original cultivar after removing the introduced TALEN gene by breeding. Focusing on the fact that genome editing can be induced via transient expression of the TALEN gene, a research group of Osaka University and RIKEN successfully obtained segregants having mutated SSR2 without the TALEN gene incorporated into potato genome (“null segregants”) (Yasumoto et al.2020). Potato, which is a vegetative reproducing crop and can propagate via corm (tuber) production, can be grown and domesticated if properties of the resulted genome-edited individual are evaluated with no problem.
Further, the group found that not only SGA synthesis but also sprouting could be controlled by creating knockdown other genes associating with SGA biosynthesis (Umemoto et al.2016). They are now developing potatoes that do not sprout during storage. A group of Tokyo University of Science has successfully developed glutinous potato with low amylose content by conducting genome editing targeting the potato starch synthase (GBSS) gene using CRISPR/Cas9 systems combined with a translational enhancer dMac3 (Kusano et al.2018). Development of a new cultivar of potato with a new glutinous texture is awaited.
Led by the researchers described above, the “Liaison Council of New Technology for Potatoes” was established in July, 2018, where researchers and relevant parties and companies in the potato industry are to associate and hold discussion on development of new cultivars of potato by genome editing aiming to facilitate the effort to their commercialization.
(3) Rice (Oryza sativa)
As rice is often used as a model plant for research, there may be many examples in which genome editing is used for analyses of its genetic functions or it is used as a material for developing genome editing technologies. As an example of research and development of rice with practical traits, a group led by NARO is working on the development of “sink-capacity modified rice for super high yield” (Komatsu 2018). While it is said that domestic production of rice (Oryza sativa) is sufficient, rice for business use, which is used in the food service and restaurant industries, is getting insufficient. The sink-capacity modified rice is under development aiming at reducing production costs by increasing yields, with a view to using it for business and feed purposes.
The term “sink capacity” refers to the ability of plants to store photosynthetic products (starch) in spikes and other parts of the plant. Two lines have been developed: one intends to increase the number of grains by modifying Gn1a (a cytokinin oxidase gene), which controls the branching of rice spikes, and the other to increase grain weight by modifying TGW6 (an indoleacetic acid glucosidase gene), which regulates grain length and weight. The line with modified Gn1a is created with two separate methods: one uses a regular CRISPR/Cas9 system, and the other uses “Target-AID” developed by Professor Nishida of Kobe University (a technique to replace C to T in a base at the target site using a combination of CRISPR/Cas9 and lamprey-derived cytidine deaminase) (Nishida et al.2016). Both methods turn off the function of Gn1a by displacing reading frames of codons or generating stop codons, leading to the target trait to be expressed.
A field cultivation test of sink-capacity modified rice was started from FY2017 in the isolated field of NARO (Tsukuba, Ibaraki) (NARO, 2017). At that time, rules for handling genome edited crops were not yet established, and the cultivation tests were conducted after obtaining approval from the Minister of the Environment and the Minister of Education, Culture, Sports, Science and Technology for the type-1 use of living modified organisms. This was the first field cultivation test in Japan for genome edited crops. In this test, traits of the rice including yields, forms of spikes, and sizes of rice grains are examined, and selection of lines is ongoing.
In addition, NARO is developing rice with enriched content of oleic acid, which is said to be good for health for its resistance to oxidization, using a knockout gene of FAD2, an enzyme which catalyzes desaturation of fatty acids, using genome editing (Abe et al.2018)。
A research group of Okayama University and NARO has developed wheat with “resistance to pre-harvest sprouting” using a CRISPR/Cas9 system (Abe et al.2019). In Japan, the resistance to pre-harvest sprouting of grains on spikes is an important trait in breeding of wheat species as their spikes emerge during the rainy season. However, cultivars and genetic resources with this trait have been few at present.
This research was started from the discovery of Qsd1, a gene involved in barley seed dormancy, by the group of Okayama University and NARO (Sato et al.2016). When barley loses the function of Qsd1, the period of its seed dormancy prolongs. The research group first identified three Qsd1 homologous genes in wheat, one from each of its three genomes, then compared their sequences and applied genome editing targeting a common sequence. The result showed that dormancy was prolonged and germination from spikes was suppressed only when all three Qsd1s were mutated. Those findings in wheat, which is hexaploid, showed the effectiveness of genome editing in breeding hyperploid crops, in the same way as the potato described above. It also demonstrated usability of the method for modifying wheat genes using the genetic information elucidated for barley that is diploid.
Because the wheat that the research group used for genome editing in this study is a foreign variety called “Filter,” which is suitable for tissue culture and genetic modification, it will require backcrossing and other handlings to develop wheat varieties resistant to pre-harvest sprouting of grains on spikes for use in Japan.
On the other hand, another group of the NARO and Kaneka Corporation has developed the “in planta particle bombardment method” (iPB method) to introduce DNA and other materials directly into shoot apical meristem of seeds using wheat as a material, and has also succeeded in genome editing (Hanada et al. 2018). The iPB method can be applied to the varieties that are difficult to cultivate or crops other than wheat since it does not require steps of callus culture and redifferentiation. This method is expected further to be used in a wide range of cultivars and crops.
(5) Flowering Plants
For flowering plants, Associate Professor Ono at the University of Tsukuba and his colleagues made a success in genome editing of morning glory for the first time in the world in 2017 (Watanabe et al. 2017). A “white morning glory” was produced by applying genome editing with a CRISPR/Cas9 system to the anthocyanin synthase gene, DFR-B, of a variety whose flower color is purple due to the accumulation of anthocyanins, a type of flower pigment. Morning glory was said to be brought from China in the Nara period, which took about 850 years for the first white morning glory to be recorded in the Edo period. With the use of genome editing technology, however, the white morning glory was created in only about one year.
In 2017, a group of NARO succeeded in genome editing in chrysanthemums (Kishi-Kaboshi et al.2017). This study showed that genome editing targeting a pre-introduced fluorescent protein gene reduced its level of fluorescence. Although its further application to practical traits of chrysanthemum is anticipated, this plant has many hurdles for breeding, such as its hyperploidy (hexaploid), vegetative reproducing nature, and genome that has not been sequenced yet. Referring to the successful examples of potatoes and wheat, technology suitable for chrysanthemum is expected to be developed.
In addition, the Iwate Biotechnology Research Center has successfully altered the flower colors of bluewings (Torenia fournieri) and gentians (Gentiana scabra) using a CRISPR/Cas9 system (Nishihara et al. 2018; Tasaki et al. 2019).
As flowering plants are not edible in general, blue carnations and roses created by genetic modification technology have been commercialized in Japan. Thus, some say that flowering plants should be given priority in the practical application of genome edited crops as it is anticipated that they will be more easily accepted by consumers. In any case, varieties of flowering plants in demand have been changing in a short time in recent years, and there are high expectations for accelerated development of new varieties. Quick breeding through genome editing technology is awaited.
(6) Fruit Trees
Since fruit trees (and trees in general) inherently take long time to breed, acceleration of breeding by genome editing is expected. In Japan, technologies for application to apples and grapes are under development.
A research group consisting of Tokushima University, NARO and others have made a success in genome editing in apples for the first time in the world in 2016 (Nishitani et al.2016). The efficiency in which mutants were obtained then reached up to 14 % among the CRISPR/Cas9 transformed individuals. The target is a gene, PDS, encoding a carotenoid biosynthetase, which is a model target to visually analyze introduction of mutation rather than traits useful for agriculture. Application to create practical traits such as self-compatibility is expected in the future.
A group of NARO also succeeded in genome editing in grapes in 2017, targeting the PDS gene (Nakajima et al. 2017). In addition, research is ongoing to change the skin color to produce “Red Shine Muscat”.
Because one generation of fruit trees is long and their varieties have heterogeneous genetic compositions, it is not practical to remove introduced foreign DNA by cross-breeding. Genome editing technology that does not require introduction of foreign DNA for fruit trees needs to be established in the future.
A group led by Assistant Professor Kinoshita at Kyoto University has been working on genome editing in red sea bream (in collaboration with Kindai University) and tiger pufferfish (in collaboration with the Japan Fisheries Research and Education Agency). The representative breeds with the intended trait are “fleshy red sea bream and tiger pufferfish” with increased muscle mass. Inspired by the spontaneous mutation of the myostatin gene (involved in the control of muscle mass) known in cattle, genome editing of the myostatin gene was performed in red sea bream and tiger pufferfish, and an increase in muscle mass (edible portion) of about 20% was observed in red sea bream (kishimata et al. 2018). In this study, genome editing was performed by directly injecting mRNA or RNP into fertilized fish eggs, rather than through temporary genetic recombination as in plants. The third and subsequent generations of genome edited strains have been obtained for red sea bream, and their practical application is under preparation now.
The Japan Fisheries Research and Education Agency is developing “slow-swimming tuna” suitable for aquaculture (Higuchi et al.2019). Similarly, Kyushu University is developing mackerel with less aggression by genome editing to improve their suitability for aquaculture. There are growing concerns about the depletion of natural fishery resources including tuna, so that a global shift from fishing to aquaculture is anticipated. The use of genome editing is expected to increase the productivity of aquaculture.
Developments of other genome-edited products including broccoli (self-compatibility), cedar (pollenless cedar), koji-mold (Katayama et al. 2016), cricket (Watanabe et al. 2012), and chicken (eggs) (Oishi et al. 2016) are underway in Japan.
Development of Genome Edited Agricultural, Forestry and Fishery Products Overseas
The genome edited crop whose commercialization was first announced in the world was glutinous corn or waxy corn developed by DuPont (current Corteva Agriscience) (Corteva Agriscience 2016). At the time of the announcement in 2016, it was scheduled to be “commercialized in 5 years”, which is going to be postponed.
For the moment, the only genome edited food that has been in practical use in the world is “High Oleic Soybean” developed by Calyxt (Calyxt, Inc.2019). High oleic soybean oil extracted from soybeans whose FAD2 gene is modified as explained in the section, (3) Rice, is on the market in the U.S.
Further, developments of other genome edited agricultural, forestry and fishery products have been reported, including mushrooms that do not turn brownish, cattle without horns, wheat resistant to powdery mildew, tilapia (freshwater fish) with high growth efficiency, wheat containing increased dietary fiber, romaine lettuce that does not turn brownish, and cacao adapted for climate change.
Future Directions — Where Should We Go? —
(1) Development of Genome Edited Crops Appreciated and Utilized by Stakeholders
As you can see from the trends in the development of genome edited crops explained above, genome editing technology is utilized in the ongoing development of crops (cultivars) that will solve the issues and meet the needs of various stakeholders (consumers, food suppliers and processors, producers, etc.). As the issues and needs stakeholders have are not few in the first place, it is essential to have them experience various benefits this technology provides, for their understanding and acceptance of genome edited crops.
This seems to be in contrast to the situation of genetic modification technology which (at least to the extent it is used commercially) mainly makes use of traits that benefit producers, such as resistance to herbicide and pest. To make sure to deliver various benefits of genome edited crops to consumers, it is important to foster an environment that facilitates the widespread use of this technology in the public including small and medium-sized companies such as community-based seed and seedling manufacturers, by setting the regulations (handling rules) for genome edited crops not to be excessively strict.
To deepen consumers’ understanding of genome edited crops and foods, it is greatly significant to develop cultivars and foods that consumers will experience benefits directly, and to proceed their commercialization. Many companies are keeping a wait-and-see attitude toward genome-edited crops because they concern that the crops may not be widely accepted by consumers. However, consumers will not deepen understanding of their value unless they pick up and appreciate the crops. The GABA-rich tomato expected to be first marketed in Japan can contribute to maintaining consumers’ good health, and people are interested in seeing how it is accepted in the market. While some consumers may appreciate GABA’s antihypertensive or relaxing effects, others have little interest in it. It is expected that various crops and foods will be commercialized continuously besides GABA-rich tomato in response to the various values of consumers.
On the other hand, for food producers, suppliers and processors, the practical application of genome edited crops should be explained thoroughly from the view point of what kind of issues it solves and how it supports agriculture and industry in Japan in order to acquire empathy of consumers. For example, if we fully explain that the improvement of a long shelf life and parthenocarpy will contribute to reducing the workload of farmers and thus protect Japan’s agriculture, we may be able to gain the consumers’ understanding. In recent years, “ethical consumption”, consuming behavior in consideration of impacts on the environment, has been drawing interests, so that explanation in light of this ethical consumption will be required.
For further development of genome edited crops, it is required to set goals and development methods that envision the fulfillment of the accountability and acquisition of consumers’ understanding. To do so, breeders, seed and seedling manufacturers and lab researchers should be integrated more strongly to proceed the research and development. To provide agricultural products and foods which meet needs of stakeholders timely, it is significant to collect information of the latest technological development and shorten the period for development by introducing necessary techniques as soon as possible.
(2) Directions of Development of Crops Using Genome Editing Technology
Although there may be various opinions on what crops should be prioritized for development using genome editing technology, it is basically considered as a useful technology that can rapidly develop varieties that will solve various problems and meet the needs of the agricultural sector.
One direction of development attracting interests recently is “health functionality”. Japan’s national health care expenditures exceed 42 trillion yen per year, and its burden on the national budget is increasing, making control of these expenditures an urgent issue. As a countermeasure to this problem, “promotion of health through food” is being promoted, and the health functional components of agricultural, forestry, and marine products and foods are being focused. If the synthetic pathways of these components are elucidated, it is possible to develop crops containing increased amounts of functional components through genome editing, as GABA-rich tomatoes and high oleic soybeans have been developed actually.
Another direction is to realize “sustainable society” or to adapt to “SDGs”. In the context of global environmental changes such as climate warming, response to SDGs (sustainable development goals) has become an international subject. In Japan, the “Sustainable Development Goals (SDGs) Promotion Headquarters” formulated the “SDGs Action Plan”. In the “Bio-Strategy 2019” decided in July, 2019, realization of the sustainable sound material-cycle society is claimed. The goal of breeding to contribute to this direction may include, for example, its use for improvement of long-life crops that will reduce the amount of food waste and crops for biomass resources that will be materials for bioplastics and biofuels.
(3) Conclusion — Using the Latest Technology to Enrich Our Lives —
For the utilization and distribution of agricultural, forestry and fishery products developed with genome edition, the public understanding and recognition that agricultural products have “breeds” and our rich diet is based on the results of selective breeding should be spread widely. To effectively utilize the technology that human beings have acquired at last, it will be important to use it with consideration for safety and communication among relevant people from researchers to consumers.
Figure-1 Applications of genome edition to various crops
(Upper Panels) from upper left: an experimental line of GABA-rich tomato (courtesy of Prof. Ezura of the University of Tsukuba), potato containing almost no natural toxin (courtesy of Prof. Muranaka of Osaka University), rice with high-sink capacity (courtesy of Dr. Komatsu of NARO)
(Lower Panels) from lower left: fleshy red sea bream (courtesy of Assistant Prof. Kinoshita of Kyoto University and Prof. Iedo of Kindai University), white morning glory (courtesy of Associate Prof. Ono of the University of Tsukuba), wheat with resistance to pre-harvest sprouting of grains on spikes (courtesy of Dr. Abe of NARO)