Benefits of artificially cultured meat

On June 23, 2021, Future Meat Technologies, based in Rehovot, Israel, announced the opening of the world’s first artificial cultured meat plant [1]. The plant can produce 500 kg of cultured meat per day, equivalent to 5,000 hamburgers, according to the company’s CEO, Rom Kushuk. Moreover, the plant is remarkable for its capacity to generate meat with significantly lower greenhouse gas emissions, land use, and freshwater consumption compared to traditional livestock farms. It is anticipated to address environmental issues related to meat production. This article focuses on lab-grown meat, explaining what it is and how genome editing is applied in its research.

What is artificially cultured meat?

Artificially cultured meat, also known as cultured meat or clean meat, is meat that has been artificially produced. It is defined as muscle cells extracted from a living animal with minimal invasion, nourished, and cultured to develop into muscle tissue. This tissue is biologically identical to the meat tissue that is the main component of the meat we normally eat. In other words, artificially cultured meat does not create meat components from scratch but instead artificially cultivates animal muscle cells outside the animal’s body to a size that can be eaten by humans. So, what are the benefits of artificially producing meat using this method?

Artificially cultured meat is revolutionizing the meat market

Artificially cultured meat has the advantage of maintaining an environmentally friendly supply to meet the rising demand for meat. The global population, especially in developing nations, is projected to reach 8.5 billion by 2030 and nearly 10 billion by 2050 [2]. In response to this trend, there is a steady increase in demand for meat. It is expected that annual global meat consumption will climb from approximately 317 million tonnes in 2016 to surpass 500 million tonnes by 2050 [3][4]. To meet the growing demand for meat, humans have tried to increase productivity by breeding livestock and improving breeding methods and feed. However, meat production requires significant resources. According to a 2010 study, the water footprint for producing 1 kg of meat is much higher than that for vegetables and fruit, especially beef, which requires 15,415 liters of water [5]. In addition, livestock production requires not only water but also significant amounts of land and fuel for transportation. Even though an estimated 130 million birds and 4 million pigs are slaughtered for meat every day worldwide, it generates a significant amount of food waste at the point of consumption. Against this backdrop, artificially cultured meat has gained attention in recent years as an alternative to conventional meat. Artificially cultured meat offers several advantages:

(i) efficient use of space and resources, with low water and feed requirements;
(ii) elimination of the need for animal slaughter;
(iii) reduced emissions of harmful substances such as CO2 and methane that are typically associated with livestock production.

Producing meat closer to the place of consumption can reduce the amount of toxic substances produced during meat production, processing, and transport, as well as the amount of methane emitted by cattle. Artificially cultured meat has the potential to overcome many problems in conventional meat production. Some foreign countries have already granted marketing authorization for artificially cultured meat.

Artificially cultured meat and CRISPR

Here, we would like to introduce an example of how genome editing technology, particularly CRISPR/Cas9, is used in artificially cultured meat. CRISPR/Cas9 is the current leading technology for genome editing, developed by Emmanuelle Charpentier and Jennifer A. Doudna. CRISPR/Cas9, which is called a third-generation genome editing tool following TALEN and ZFN, manipulates trait expression by cutting the target DNA sequence-specifically and deleting (knocking out) the target gene. Furthermore, by utilizing the repair mechanism that accompanies DNA breaks, it is also possible to express the desired gene by introducing donor DNA from outside. As previously mentioned, artificial cultured meat is based on animal muscle cells and tissue culture. Muscle cells can be cultured by providing them with growth factors that promote cell proliferation and differentiation, as well as a favorable medium for cell growth. The challenge in producing artificially cultured meat is how to enable rapid cell growth.

As an answer, there are two possibilities:
① Accelerate the growth rate of muscle cells themselves ← “Improve the internal mechanism for growth”
② Improving growth factors and culture media to be more efficient ← in “Providing a desirable environment”

Several companies are working on each method to achieve these two goals using genome editing technology using CRISPR/Cas9, and I will introduce one of them one by one.

UPSIDE Foods (formerly Memphis Meats)

UPSIDE Foods is a startup established in 2015 by cardiac surgeon Uma Valletti and biologist Nicholas Genovese [6]. The company is based in San Francisco, California, USA. UPSIDE Foods’ technology utilizes CRISPR/Cas9 to modify muscle cells and produce artificially cultured meat. According to documents from the Patent Cooperation Treaty [7][8], CRISPR/Cas9 is used to inactivate the ‘Rb gene’ in muscle cells, which arrests the cell cycle. This promotes cell cycle progression and also inactivates the ‘p15 gene’ and ‘p16 gene’, which suppress the expression of telomerase, a protein involved in cell life extension. The artificial cultured meat produced by UPSIDE Foods can be classified as one of Japan’s ‘genome-edited foods’ because it does not involve the insertion of genes.

Core Biogenesis

Core Biogenesis is a French startup founded in 2020 by two young researchers, Alexandre Reeber and Chouaib Meziad, with three platforms: cell therapy for neurodegenerative diseases, development of growth factors for cultured meat production and mRNA production, and development of enzymes for mRNA production [9]. The cultured meat industry has taken notice of them because they edit the genome of the growth factors in the culture medium, not the muscle cells they cultivate. According to research, growth factors are the most expensive media used for cultured meat growth [10]. However, by adapting CRISPR/Cas9 genome editing to plants that produce growth factors, they can be produced 25 times more efficiently than before [11]. This means that CRISPR/Cas9 genome editing of growth factor-producing plants can reduce the cost of cultured meat production to one-tenth of conventional methods [11].

The Future of Artificially Cultured Meat

While Japan may not experience the same meat-related problems due to its abundance of food, a broader perspective reveals that food problems associated with population growth are much larger and deeper-rooted than previously thought. Numerous researchers and companies are addressing these issues using the latest technologies. As members of the global community, we Japanese need to take an interest in these issues and consider how to solve them.

References

[1] Company opens the first industrial cultured meat facility, with immediate outlook toward U.S. expansion
[2] 世界の人口推計2019年版
[3] What is the true cost of eating meat?
[4] Hannah Ritchie, “Half of the world’s habitable land is used for agriculture “, Our World in Data, Nov. 11, 2019.
[5] Mekonnen, Mesfin & Hoekstra, Arjen. (2010). The green, blue and grey water footprint of farm animals and animal products. American Journal of Hematology – AMER J HEMATOL.
[6] UPSIDE Foods
[7] Method for scalable skeletal muscle lineage specification and cultivation
[8] Methods for extending the replicative capacity of somatic cells during an ex vivo cultivation process
[9] Core Biogenesis
[10] Growth factor research is key to making cell-based meat affordable
[11] Using plants as biofactories to produce high-value molecules 10 times cheaper

The post Cultured meat is changing the world⁉-Genome editing and the future of meat first appeared on Setsuro Breeding - 生物の品種改良を精密・高速に実施する精密育種サービス.

The Birth of the Genome-Edited Tomato

Japan’s inaugural genome-edited food is a tomato, a high-value-added crop resulting from collaborative research between the University of Tsukuba and Sanatech Seed Co. Ltd., a startup affiliated with the university. Dubbed “Sicilian Rouge High Gaba,” this tomato received notification approval from the Ministry of Health, Labor, and Welfare in December 2020 [1].

One reason genome-edited foods have garnered attention is the implementation of the notification system [2], initiated in October 2019. Unlike genetically modified foods, which integrate genes from foreign species, genome-edited foods target trait alterations by selectively knocking out specific genes within the genome.

The Ministry of Health, Labor and Welfare suggests the following. ‘If the level of edited off-target genetic mutation is difficult to judge compared to mutations that could also occur stochastically in nature, and if the transgene does not remain, it is reasonable to treat it differently from GM food.’

Starting with Sanatech Seed tomatoes, the future of genome-edited foods on our tables is not far off. Eventually, we may be eating genome-edited foods as a matter of course (or even without knowing that they are genome-edited).

Issues in Plant Genome Editing as Seen in Review Papers

① The need to reconsider regulations

We commence with a review paper entitled “Gene Editing in Plants: Progress and Challenges” authored by Yanfeni Mao and co-authors [3], which was submitted to the National Science Review in January 2019. Yanfeni et al observe that genome editing research in plants is steadily advancing, thanks to the emergence of CRISPR/Cas9 and CRISPR/Cas12a.

Simultaneously, several obstacles hinder the utilization of genome editing in crop breeding. Foremost among these is the ongoing discourse regarding the classification of genome-edited crops as genetically modified organisms (GMOs). In Japan, as previously noted, regulatory measures have been established since October 2019, delineating standards for genome-edited crops and GMOs, thereby distinguishing between the two. Similar determinations have been made in the United States, where genomic research is flourishing. Conversely, in Europe, genome-edited crops face stringent regulations as they are equated with GMOs. Furthermore, disparities in regional attitudes frequently result in import and export limitations, posing research impediments. Given that numerous crops are traded internationally, these restrictions can wield considerable influence.

Yanfeni argues that concerns about treating genome-edited crops are unfounded and that regulations should be re-examined. This is because most genetic mutations induced by CRISPR are small insertions or deletions (indels), rather than large insertions or rearrangements of gene fragments. These mutations are often found in plants grown under natural conditions. They argue that genome-edited crops should be treated like conventional crops because they are common in plants and can be induced on a large scale using radiation or chemical mutagens, just as Japan has done.

Another important issue to consider, in addition to regulatory concerns, is whether society will accept genome-edited crops. Despite the legality of genome editing, the mere mention of it often evokes unfounded fears of science fiction scenarios. While genome-edited foods are a technical revolution, their acceptance by the general public is crucial for their success. Communicating the benefits of genome-edited foods to non-scientific audiences will be a key challenge in the future.

②Off-target Effects

The following technical challenge concerns off-target effects. These effects occur when the gRNA sequence mismatch tolerance in CRISPR/Cas9 and other factors cause cleavage of a different target than intended, resulting in irreversible genetic mutations. In recent years, machine learning and deep learning tools have emerged to predict the target gRNA more accurately. However, these tools still face many challenges, and the off-target effect has not been fully resolved.

However, Yanfeni et al claim that off-target effects do not necessarily hurt crops; some off-target effects may lead to mutations that are desirable to us or may not pose any particular problems. In other words, such reproductive stocks should be retained, and only those individuals who have developed undesirable mutations should be eliminated.

The same is detailed in the 2020 paper ‘Plant Genome Editing and the Relevance of Off-Target Changes'[6] by Nathaniel Graham et al.

According to Nathaniel and his colleagues, many plants develop multiple independent reproductive organs within a single organism and thus have a smaller impact on the entire population than the off-target effects that occur in animals. Therefore, it is suggested that selecting individual plants with useful phenotypes through proper management can minimize the impact of off-target effects.

However, this claim may be sophistry the issue as these mutations are not always visible. Efforts will continue to develop sequence-finding tools without off-target effects and appropriate CRISPR/Cas vectors.

③ Impact of genome editing on surrounding plants

Finally, let’s consider new problems that may arise from genome editing. Finally, let’s consider new problems that may arise from genome editing. In the article ‘Herbicide Resistance: Another Hot Agronomic Trait for Plant Genome Editing’ by Amjad Hussain and colleagues [7], published in Plants in 2021, The authors suggest that the rise of genome-edited crops using CRISPR/Cas could lead to changes in surrounding weeds during cultivation.

Weeds compete with crops for water and nutrients, which can negatively impact production. Additionally, weeds can serve as parasitic sites for pathogens and insects, which can infect crops and harm native ecosystems. Herbicides are often used to control weeds when cultivating crops. However, non-selective herbicides such as glyphosate and paraquat can also harm the crop.

To address this issue, genome-edited crops that are tolerant to herbicides have been developed in recent years. Research has already been published on the development of herbicide-tolerant crops in over 10 species, including rice, wheat, and watermelon, according to Amjad Hussain. If genome editing of crops can result in herbicide tolerance as a side effect, it may seem like a reasonable approach.

However, the development of herbicide-tolerant crops may lead to increased herbicide use, which can raise costs and reduce effectiveness as weeds become resistant. This creates an ironic situation where genome editing research on herbicide resistance may lead to increased herbicide use and decreased effectiveness.

Although genome editing technology is revolutionary, it poses many challenges. Focusing solely on the advantages can cause problems and may even exacerbate the situation. Therefore, careful consideration and attention are needed to make beneficial use of this technology.

This time based on three review articles, We have introduced three points worth paying attention to. Furthermore, ongoing research on plant genome editing highlights many other issues that need to be resolved as the technology progresses. It is important to maintain interest and discussion on genome-edited foods to improve our lives in the future.

References

[1] 日本経済新聞 「「ゲノム編集食品」国が初承認　トマト流通へ」
[2] 薬事・食品衛生審議会食品衛生分科会 新開発食品調査部会 報告書 「ゲノム編集技術を利用して得られた食品等の食品衛生上の取扱いについて 平成31年3月27日 」
[3] Yanfei Mao, Jose Ramon Botella, Yaoguang Liu, Jian-Kang Zhu, Gene editing in plants: progress and challenges, National Science Review, Volume 6, Issue 3, May 2019, Pages 421–437, https://doi.org/10.1093/nsr/nwz005
[4] Pallarès Masmitjà M, Knödlseder N, Güell M. CRISPR-gRNA Design. Methods Mol Biol. 2019;1961:3-11. doi:10.1007/978-1-4939-9170-9_1
[5] Lin Y, Cradick TJ, Brown MT, et al. CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res. 2014;42(11):7473-7485. doi:10.1093/nar/gku402
[6] Graham N, Patil GB, Bubeck DM, et al. Plant Genome Editing and the Relevance of Off-Target Changes. Plant Physiol. 2020;183(4):1453-1471. doi:10.1104/pp.19.01194
[7] Zhang, R.; Liu, J.; Chai, Z.; Chen, S.; Bai, Y.; Zong, Y.; Chen, K.; Li, J.; Jiang, L.; Gao, C. Generation of herbicide tolerance traits and a new selectable marker in wheat using base editing. Nat. Plants 2019, 5, 480–485.

The post Will Plant Genome Editing Benefit Us？ – Hopes and Challenges of Genome-Edited Crops as Seen in Review Papers from Around the World – first appeared on Setsuro Breeding - 生物の品種改良を精密・高速に実施する精密育種サービス.

Threat of Infectious Diseases

Since the confirmation of a cluster infection in Wuhan, China in 2019, the novel coronavirus infection, known as COVID-19, has caused a global pandemic. COVID-19 can be considered one of the most notorious infectious diseases in human history, significantly altering our lives in ways unparalleled before.

However, infectious diseases posing a threat to humanity are not a novel occurrence. Throughout history, diseases like smallpox, which caused numerous deaths since ancient times, the Black Death that claimed one-third of Europe’s population in the 14th century, and the Spanish flu that spread after World War I. Human history has always been a battle against infectious diseases. Among them, “Malaria infection” is known as an infectious disease that has continued to claim numerous lives as a zoonotic infection from prehistoric times, affecting both humans and animals. Even in modern times, people continue to lose their lives due to malaria infections. So this time, the discussion will approach attempts at treating malaria from the standpoint of genome editing. About Malaria

Before delving into the discussion of malaria treatment using genome editing, let’s first organize information about malaria. Malaria is a bloodborne infectious disease transmitted by mosquitoes of the Anopheles genus, carrying parasites known as Plasmodium spp[1]. There are five types of Plasmodium parasites that infect humans: P. falciparum,　P. vivax, P. malariae, P. ovale, and P. simian. Plasmodium falciparum, causing tropical malaria, is particularly known as “malignant malaria” and poses a higher risk of severe complications.

The malaria parasite primarily infects red blood cells. Infected red blood cells are destroyed due to the activity of the malaria parasite. As a result, there is an increased burden on the spleen, which serves as the waste disposal site for red blood cells, causing it to swell significantly. Additionally, blood clots, referred to as “thrombi,” can form, blocking blood vessels in various organs and impairing their function. Among these complications, cerebral malaria, which affects the brain, is particularly lethal. Patients who develop cerebral malaria often fall into a coma and ultimately succumb to death.

Therapeutic drugs for malaria infection have been developed and put into practical use. For example, quinine is a medication derived from substances found in the bark of the cinchona tree and is known to be the precursor to major antimalarial drugs such as chloroquine and primaquine. Additionally, artemisinin is a medication derived from the sweet wormwood plant (Artemisia annua), and the Chinese researcher Tu You-you, who discovered it, received the Nobel Prize in Physiology or Medicine in 2015 [2].

Humanity has been developing various therapeutic drugs to eradicate malaria infections. However, considering the current situation where over 600,000 people still lose their lives to malaria infections worldwide every year, it is believed that an approach solely based on conventional drug development may find it challenging to eliminate malaria infections. Therefore, this time will introduce some research efforts utilizing CRISPR/Cas9 to address malaria infections.

Genome Editing and Malaria

Mainly now CRISPR/Cas9, a genome editing technology that is currently primarily utilized, was first applied to malaria parasites in the world through the research conducted by Mehdi Ghorbal and colleagues, published in Nature Biotechnology in 2014 [3]. Mehdi and his team demonstrated that CRISPR/Cas9 could induce double-strand breaks in malaria parasites with ease, given the appropriate design of guide RNA (gRNA). Furthermore, they confirmed the ability to introduce foreign genes into malaria parasites by utilizing homologous recombination with the introduction of donor DNA.

In this study, Targeting the egfp region of the malaria parasite’s chromosome, double-strand breaks were induced. As a result, resistance genes against folate antagonist metabolism inhibitors in humans were introduced. Successful reproduction of malaria parasites lacking egfp and possessing resistance to folate antagonist metabolism inhibitors was achieved within approximately three weeks. In all of these experiments, no significant off-target effects were observed. (Off-target effects refer to unintended genetic mutations that may occur in the context of CRISPR/Cas9.) With the insights gained from these studies, it is anticipated that research on the pressing issue of drug resistance in malaria parasites will continue to advance by through the judicious use of CRISPR/Cas9.

Andrea and his team aimed to create mosquitoes that have lost reproductive function by knocking down the intron 4–exon 5 regions of the doublesex gene, involved in mosquito reproduction, using CRISPR/Cas9. Consequently, the genome-edited mosquitoes in the cage areas, confined in cages, lost reproductive function in nearly all individuals by the 7th generation, ultimately leading to the extinction of the mosquito population.

While this experiment was confined to cages, if these genome-edited mosquitoes were released into the actual natural environment, it could result in the loss of reproductive function in mosquitoes worldwide, potentially leading to the extinction of the mosquito population. The extinction of the vector for malaria parasites would effectively mean the eradication of malaria infections.

From the perspective of eradicating malaria, the use of CRISPR/Cas9 for gene drives can be considered beneficial for humanity. However, further research is needed to understand how the extinction of mosquitoes, such as Anopheles, through CRISPR/Cas9-driven gene drives, would ultimately impact humanity. Ethical considerations surrounding the deliberate extinction of populations through artificial gene drives also require careful discussion. Interference with ecosystems through casual genome editing should be approached with caution.

Furthermore, not only for malaria but also for pandemics that occur approximately every ten years, such as SARS, new influenza, and COVID-19, The use of CRISPR/Cas9 technology may be effective. Even as COVID-19, which has spread at the fastest rate in recorded history, begins to subside, it is not difficult to imagine the emergence of different infectious diseases in the future. Taking the lessons from this pandemic, it is hoped that humanity, leveraging various technologies, including the wisdom of CRISPR/Cas9, will carefully consider the balance between eradicating infectious diseases and their impact on ecosystems. The optimal use of CRISPR/Cas9 for the benefit of humanity and the Earth is desired.

References

[1] World Health Organization. “Malaria”.
[2] The Nobel Foundation. “Press release: The Nobel Prize in Physiology or Medicine 2015.” The Nobel Prize. 15 October, 2020.
[3] Ghorbal, M., Gorman, M., Macpherson, C. et al. Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system. Nat Biotechnol 32, 819–821 (2014).
[4] Ariey, F., Witkowski, B., Amaratunga, C. et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature 505, 50–55 (2014).
[5] Kyrou, K., Hammond, A., Galizi, R. et al. A CRISPR–Cas9 gene drive targeting doublesexcauses complete population suppression in caged Anopheles gambiae mosquitoes. Nat Biotechnol 36, 1062–1066 (2018).

The post Genome Editing to Protect Humanity from Infectious Diseases – The Battle of CRISPR/Cas9 and Malaria first appeared on Setsuro Breeding - 生物の品種改良を精密・高速に実施する精密育種サービス.