Download PDF Summer Bridge on Smart Agriculture June 15, 2022 Volume 52 Issue 2 People everywhere rely on agriculture in one form or another – for food, animal feed, fiber, and other necessities. The summer 2022 articles describe precision indoor farming and alternative protein food systems, advances in food processing, genome editing, digitalization, sustainable and regenerative agriculture, the role of a circular economy, and the important role of policy. Engineering Genomes for Sustainable Agriculture: Opportunities and Challenges Tuesday, June 14, 2022 Author: Rodolphe Barrangou Genome engineering is enabling the design of enhanced organisms such as crops, livestock, and trees to promote sustainability. The realm of biology and genetics is in the midst of a genome editing revolution, fueled by CRISPR-based technologies for manipulating and engineering the DNA blueprint of virtually all organisms in the tree of life. These molecular machines are derived from the adaptive immune systems in bacteria, which consists of clustered regularly interspaced short palindromic repeats (CRISPR) and their associated effectors (Cas) (Barrangou et al. 2007). Building on the clinical success of CRISPR-based gene therapies, breeders are rapidly deploying genome editing in research and development activities, seeding commercial pipelines. Given the tremendous potential of genome engineering for agricultural applications, it is essential to determine the role and implementation of genome engineering as part of the solution for a sustainable agricultural system. CRISPR Basics: Engineering Genomes with Molecular Machines The genomes of every organism are composed of a series of four letters, A-T-C-G, arranged in the mesmerizing double helix whose complementary strands encode signals for genes (DNA sequences). These sequences are expressed into messenger molecules (RNA transcripts) that are in turn translated into effectors (proteins with physical and chemical attributes). Over the past 2 decades, the world of genetics has been revolutionized by the ability to both read genomes, transcriptomes, and proteomes at very high scale and manipulate them. Genome editing now enables molecular engineers to rewrite selected portions of the genome of virtually any species. FIGURE 1 Genome engineering basics. Top left: DNA is transcribed into RNA, which is then translated into proteins. Middle and bottom left: Using CRISPR, DNA molecules can be cleaved at programmable locations to trigger repair pathways that can be coopted to generate selected mutations, insertions, or deletions in the genome for desired outcomes. Right: Versions of CRISPR effectors tethered to transcriptional effectors can control transcription by turning it on or off or modulating the level of gene expression up or down, enabling fine-tuning of the amount of a protein and the activity of the encoded protein in the host. CRISPR-Cas systems enable immunity by picking up DNA sequences from invaders (e.g., viruses) and using them as sequences (DNA “mugshots”) that drive the specific targeting and cleavage of matching elements with molecular scalpels (the Cas effectors) (Makarova et al. 2019). A decade ago, these bacterial tools were repurposed as programmable molecular machines in a biochemical advance worthy of the 2020 Nobel Prize in Chemistry for their inventors (Jinek et al. 2012). This tool was subsequently deployed to cut the genomes of human cells and enable editing precisely at the site of cleavage (figure 1) (Cong et al. 2013). By placing the CRISPR cursor at a particular location in the genome sequence, geneticists can rewrite the DNA code precisely at that position to engineer genomes through the insertion and/or deletion of single letters or whole chapters in a DNA blueprint. Mechanistically, once the chromosome is cleaved at the intended location, the DNA is patched through various naturally occurring repair pathways. Versions of these tools fused with other effectors and enzymatic machinery allow genome engineers to also modulate the strength of the signal (turn on/off, up/down the level of transcription; figure 1). If the genome were a series of letters in text, the content of a book could be edited and its oratory delivery guided. If the genome were a series of notes in a score, it would be possible to change the chords and tune the strength with which each note is played. While we shall refrain from editing Sir William Shakespeare’s sonnets or tinkering with Wolfgang Amadeus Mozart’s scores, typos in books and operas can now be corrected. There are many practical benefits, notably efficiency, precision, affordability, and convenience of deployment, although improvements are needed in the control and prediction of repair outcomes and in delivery modalities to increase penetrance of effectors in larger amounts of cellular material. Amazingly, barely a decade into the CRISPR craze, this disruptive technology has already enabled the medical community to correct DNA typos in the genomes of patients with genetic diseases. Individuals with debilitating diseases caused by inherited faulty genetic signals have had their genomes recoded at the affected locus by CRISPR engineers, with supraclinical therapeutic benefits (Gillmore et al. 2021); for example, FDA-enabled clinical trials have recently reported success for sickle cell disease (Frangoul et al. 2021). This opens intriguing opportunities beyond translational biotechnology.… Recoding Agriculture: From Sustainable Crops to a Healthier Forest The ability to engineer genomes and tinker with DNA sequences with unprecedented ease, speed, and scale is inspiring breeders of all biological entities. Genome engineers have deployed CRISPR tools in species from viruses and bacteria to plants and trees (whose genome can be 10 times larger than the human genome), including species used in food and agriculture (Zhu et al. 2020). Importantly, besides the practical advantages of this molecular “Swiss Army knife,” benevolent repositories provide open access to best-in-class technologies from a collaborative network of innovators to tens of thousands of academic researchers around the globe (Huang et al. 2019). FIGURE 2 Diverse realms of genome editing in agriculture. Top: Plants and crops such as rice, wheat, corn, and soy; trees; and fruits and vegetables such as mushrooms and tomatoes. Bottom left: Livestock including cattle, swine, and poultry. Bottom right: Microbes including bacteria, yeast, and fungi. Starting small, bacteria used in food fermentations have had their genomes enhanced to optimize their functional attributes linked to the flavor and texture of fermented dairy products such as yogurt and cheese. The fact that CRISPR-Cas systems provide adaptive immunity against viruses in dairy bacteria led to the commercial launch, more than a decade ago, of bacterial starter cultures with enhanced phage immunity in industrial settings. Most fermented dairy products are now manufactured using CRISPR-enhanced starter cultures. Since then, a variety of bacteria, yeast, and fungi (figure 2) involved in the manufacturing of bioproducts has also been CRISPR enhanced to yield commercial products such as enzymes, detergents, and dietary supplements. Moving along the farm-to-fork spectrum, most commercial crops—from corn, soy, wheat, and rice to fruits and vegetables—have had their genomes altered (figure 2). Genome engineering is used to increase yield (e.g., meristem size, grain weight) and improve quality (e.g., starch and gluten content), pest resistance (e.g., to bacteria, fungi, viruses), and environmental resilience (e.g., to drought, heat, frost). For instance, nonbrowning mushrooms with extended shelf life can be generated, and tomatoes with increased amounts of gamma aminobutyric acid (GABA) to enhance brain health have been commercialized. In addition, efforts are underway to enhance nutritional value. Expanding Applications Besides edible plant species, much ongoing work aims to optimize both the biochemical composition of biomass for biofuel genesis and the traits of species such as cotton, hemp, grasses, and flowers. In an era of plant-based meats, alternative protein sources, and lab-grown foods, it seems nothing is impossible—imagination might be the only limiting factor with regards to how CRISPR can be exploited for foods. As a gauge of industry enthusiasm and commercial adoption, it is worth noting that leading agricultural players like Bayer, Corteva, BASF, and Syngenta as well as startups like Inari Ag and Pairwise Plants are actively breeding commercial crops. Livestock breeders have joined the fray, with genome engineering of main farm species such as swine (leaner bacon), poultry (CRISPR chicken), and cattle (for both meat and dairy). Swine have also been edited with a viral receptor knockout to prevent porcine reproductive and respiratory syndrome; the approach is being evaluated for regulatory approval (Burkard et al. 2017). Breeding applications include hornless cows (for more humane treatment), resistance to infectious disease (tuberculosis in cattle), and removal of viral sequences in the genome of elite commercial livestock, notably swine. The CRISPR zoo also encompasses genetically diverse species—fish (tiger-puffer and red sea bream), cats (efforts are underway to develop hypoallergenic variants), and even butterflies (wing pattern)—illustrating the ability to deploy this technology broadly. A World Economic Forum report, Technology Innovation in Accelerating Food Systems Transformation (WEF 2018), projected that genome editing for seed improvement could boost farmer income (up to $100 billion value creation), increase food production (by 5 percent), and help reduce human micronutrient deficiency (for up to 5 percent of the affected population). With rapid population growth and challenges to the global food supply chain, the use of enabling technologies for more efficient, sustainable, and resilient agriculture is critical. Historical mistakes in public relations and ethical concerns are reminders of the importance of science communication. Given climate change concerns, it is noteworthy that genome engineering of trees—whether for enhanced wood properties, tree resilience, pest management, or environmental stewardship—can yield a healthier and more sustainable forest. For example, altering lignin content and fiber composition and linkage could substantially impact the efficiency of the pulping process, which generates paper, cardboard boxes, and hygienic tissue. Lower lignin renders wood more amenable to bioprocessing, in turn reducing the amount of energy, enzymes, and chemicals needed to generate pulp. Similar approaches hold promise for fiber-based bioethanol production and wood-derived bioproducts, with DNA-free modalities (Cas effectors loaded with guide RNA, forgoing the need to use DNA constructs) that rely on delivery of ribonucleoprotein complexes directly into seed embryos that develop into non-GMO-edited trees. As genome editing matures, it is appropriate to strategically determine how these powerful tools can best be deployed and prioritized to optimize the benefits of this disruptive technology, especially for energy efficiency and resource use. Learning from Mistakes and Shortcomings The technical challenges, however difficult and problematic, may prove to be the easier part of genome engineering. Historical mistakes in public relations for GMO technology, genetic engineering regulatory hurdles, and ethical concerns about CRISPR use in humans are all reminders of the importance of science communication. The rise of CRISPR tools has been accompanied by some drama, notably with regards to intellectual property in a high-profile kerfuffle covering interference proceedings (Sherkow 2022). Likewise, the legal and ethical ramifications of the birth of genome-edited children in China have compelled the international scientific community to define and update guidance for the responsible and transparent deployment of genome editing technologies in human cells and individuals (NAM, NAS, and Royal Society 2020). Regulation The success of genome engineering technologies in medicine and biotechnology has triggered efforts in agriculture that encompass regulatory considerations. Of particular note is the Sustainable, Ecological, Consistent, Uniform, Responsible, Efficient (SECURE) rule implemented by the USDA’s Animal and Plant Health Inspection Service; it covers plants generated through techniques that use recombinant, synthetic, and/or amplified nucleic acid to modify a genome. Welcomed by the scientific community and the agriculture industry, the SECURE rule lays a foundation for other jurisdictions to adopt similar guidelines allowing some genetic edits to be exempt from the federal regulatory process for plants. Political and regulatory support is not evenly distributed, and there is some recalcitrance in a number of countries, especially in Europe, as activist groups sow antiscience bias and skepticism. Given the business implications and geopolitical stakes, there is a case to be made that it is a question of when, rather than whether, genome engineering technologies will be broadly enabled. The debates are a reminder that organizations such as the National Academies of Sciences, Engineering, and Medicine were conceived to support evidence-informed policies, and it is encouraging that members of these academies have been asked for input regarding many of these aspects. Communication for Consumer Acceptance On a practical basis, much depends on consumer acceptance of technology. Several pandemic-related aspects illustrate the challenges of antiscience bias and shortcomings in public science literacy. Thankfully, engineering disciplines benefit from historical credibility and associated feats and milestones (e.g., aviation and mechanical engineering, computers and electrical engineering). In molecular biology, lessons learned from human genome editing highlight the need to promote trust through risk mitigation and transparency. Fortunately, there are great CRISPR stories and storytellers. The chronicles of Jennifer Doudna’s Nobel journey are inspiring (see Isaacson 2021). Likewise, the societal stakes and implications have been pondered by masterful storyteller Adam Bolt in a documentary entitled Human Nature. Such compelling narratives, in combination with the promise of genome engineering technologies for agriculture and sustainability, following the path blazed by CRISPR medicines and pioneers, lay a foundation for bridging the gap between science and society. The Future Is Now Given the need to build a resilient and more sustainable food supply, and the aggressive timeline under which this needs to happen, the ability to exploit innovative technologies is critical. To ensure that by 2050 the food supply chain can support the world’s rapidly expanding population, genome engineering requires prompt market deployment, regulatory approval, and societal acceptance so the production supply chain can be managed accordingly. References Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P. 2007. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315(5819):1709–12. Burkard C, Lillico SG, Reid W, Jackson B, Mileham AJ, Ait-Ali T, Whitelaw CBA, Archibald AL. 2017. Precision engineering for PRRSV resistance in pigs: Macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function. PLoS Pathogens 13(2):e1006206 Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. 2013. Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–23. Frangoul H, Altshuler D, Cappellini MD, Chen Y-S, Domm J, Eustace BK, Foell J, de la Fuente J, Grupp S, Handgretinger SR, and 16 others. 2021. CRISPR-Cas9 gene editing for sickle cell disease and b-thalassemia. New England Journal of Medicine 384:252–60. Gillmore JD, Gane E, Taubel J, Kao J, Fontana M, Maitland ML, Seitzer J, O’Connell D, Walsh KR, Wood K, and 16 others. 2021. CRISPR-Cas9 in vivo gene editing for transthyterin amyloidosis. New England Journal of Medicine 385(6):493–502. Huang Y, Porter A, Zhang Y, Barrangou R. 2019. Collaborative networks in gene editing. Nature Biotechnology 37:1107–09. Isaacson W. 2021. The Code Breaker: Jennifer Doudna, Gene Editing, and the Future of the Human Race. New York: Simon & Schuster. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. 2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–21. Makarova KS, Wolf YI, Iranzo J, Shmakov SA, Alkhnbashi OS, Brouns SJJ, Charpentier E, Cheng D, Haft DH, Horvath P, and 16 others. 2019. Evolutionary classification of CRISPR-Cas systems: A burst of class 2 and derived variants. Nature Reviews Microbiology 18(2):67–83. NAM [National Academy of Medicine], NAS [National Academy of Sciences], and Royal Society. 2020. Heritable Human Genome Editing. Washington: National Academies Press. Sherkow JS. 2022. Immaculate conception? Priority and invention in the CRISPR patent dispute. CRISPR Journal 5(2):174–80. WEF [World Economic Forum]. 2018. Innovation with a Purpose: Technology Innovation in Accelerating Food Systems Transformation. Geneva. Zhu H, Li C, Gao C. 2020. Applications of CRISPR-Cas in agriculture and plant biotechnology. Nature Reviews Molecular Cell Biology 21(11):661–77.  “Elite” here refers to the genetic background of the animals in which the editing was done.  7 CFR Part 340 of the Plant Protection Act (85 Fed. Reg. 29790, May 18, 2020); available at https://www.govinfo.gov/content/pkg/FR-2020-05-18/html/ 2020-10638.htm. About the Author:Rodolphe Barrangou (NAE/NAS) is a distinguished professor in the Department of Food, Bioprocessing and Nutrition Sciences at North Carolina State University.