Keywords

1 Introduction

The European Union in July 2023 released a proposal entitled, ‘Proposal for a REGULATION OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL on plants obtained by certain new genomic techniques and their food and feed, and amending Regulation (EU) 2017/625’ (EFSA, 2023). The proposal states,

The European Food Safety Authority (EFSA) concluded that, as regards risks for human and animal health and the environment, there are no specific hazards linked to targeted mutagenesis or cisgenesis…Climate change and biodiversity loss have put the focus on long-term resilience of the food chain and the need to transition to more sustainable agriculture and food systems. The European Green Deal’s Farm to Fork Strategy specifically identifies new techniques, including biotechnology, that are safe for consumers and the environment and bring benefits to society as a whole, as a possible tool to increase sustainability of agri-food systems and contribute to guaranteeing food security (EFSA, 2023: 2).

This is a significant announcement, as it signals a major shift from the precautionary approach embedded in how the EU regulates products of biotechnology in agriculture. The catch-all terminology of ‘new breeding techniques’ used in the proposal covers technologies in the pipeline and future technologies that can be applied to agrifood. The motivation behind this proposal is to work towards a sustainable food system within the European Union and to meet current and future food security goals in the face of climate change and economic uncertainties. This chapter examines a sampling of new breeding techniques in the pipeline and how they are enabling other technologies and gene editing platforms. Though our primary focus is agrifood plants, we draw attention to gene editing in related research areas, such as microbes, which are components of the broader ecosystem and fundamental to sustainable agrifood systems. This chapter focuses on the category of ‘open release products’ (organisms in the form of seeds, and plants for human/animal consumption released into the environment), as they are most relevant to agrifood production. We take a deeper look at current research being conducted on orphan crops,Footnote 1 and re-wilding or ‘wide crosses’ of crops that are important agronomically and for food security.

According to the 2017 report by National Academies of Science, Engineering and Medicine (NASEM), three classes of future gene edited products are under development: platforms, contained products, and open release products (NASEM, 2017). Platforms are tools used to create other biotech products. Contained products encompass organisms in contained environments like labs. Open release products include plants, animals, microbes, and synthetic organisms that are designed to be released into the environment. We discuss each of these classes of future gene edited products.

2 Future Gene Editing Technologies: Platforms

Platforms for biotechnology—tools used in the creation of other biotech products—include ‘wet lab’ products such as enzymes, vectors, cells, sequencing prep kits and ‘dry lab’ products like computer software (e.g., Genome Analysis Toolkit). In this vein, precision breeding or ‘smart’ breeding draws from engineering, molecular biology, agricultural science, and computer science (bioinformatics) and relies heavily on dry lab products. Generally, it uses Artificial Intelligence (AI) and automation & sequencing platforms to guide genetic changes so that scientists can quickly analyze changes to assess valuable traits and possibly remove negative plant traits using data science tools. Precision breeding can tailor traits to producers’ needs, which may differ across geographic regions, climates, or soil compositions.

Having widely accessible databases of genome sequence data can help develop AI models that can efficiently and accurately predict how plants edited for agronomic traits will fare under particular biotic and abiotic stresses. For example, Newman and Furbank (2021) used the Australian National Variety Trial (ANVT) database to develop integrated machine learning to accurately predict yields and agronomic traits. The ANVT is the largest independent coordinated national field trial network in the world. There are more than 650 trials sown across 300 locations across 10 species types including barley, canola, lentil, oats, and wheat (Grain Research Development Corporation, 2023). Machine learning allows for compressive analysis of large amounts of data, such as that in the ANVT to retrieve performance-based metrics quicker than by using previous techniques (Zhang et al., 2023).

Organisms in contained environments like labs, include microbes or are synthetic based, rather than an animal or plant host. Examples include gene edited microbes in fermenters to produce chemicals, fuels, polymers, or food additives. Using gene editing on microbes is a strong area of interest (NASEM, 2017). Research is underway to discovery how gene editing can be used to develop microorganisms to control pests, weeds, improve soil quality and aid in food processing (Wesseler et al., 2022). Scientists are looking at microbes such as yeast, algae, and bacteria to produce useful gene edited chemical biofuels. For example, the International Service for the Acquisition of Agri-biotech Applications (ISAAA) reports that the Nannochloropsis species of algae accumulates large amounts of lipids through photosynthesis and these lipids have the potential to be used as feedstocks or biodiesels. Currently, the technology needs to evolve to make biodiesel from algae cost-effective on a global scale.

Examples of the application of organisms in contained environments include developing resistance in microorganisms to specific plant pathogens, microbes that improve nitrogen fixation in the soil, and alternatives to pesticides, herbicides and fungicides in agricultural production. Novel bacteria and enzymes can be used in food processing in the fermentation process, to develop plant-based proteins, and provide alternatives to fossil fuels such as biochemicals (FAO, 2022: 12). These could be important contributions to the Farm to Fork strategy in the EU and help improve soil quality in areas with depleted soil and dependency on marginal lands for subsistence. One future expectation is using gene editing to insert synthetic DNA into genomic sequences of microbes. For example, these may help improve nitrogen fixation in the soil and assist with bioremediation in contaminated soil sites (NASEM, 2017: 23).

Several of the research scientists we interviewed expressed excitement about gene editing applications on the horizon, especially gene editing’s applications to microbes. As more knowledge is accumulated about a specific genome, the abilities for gene editing to offer solutions to some of today’s problems continues to grow. As Informant 4 (an academic researcher) stated,

…[gene editing technology] is one of the many tools that researchers in the scientific community have access to and it’s really going to change and it’s already changing the way we conduct science, because it’s combined with all the genomic information we have about the plants of interest…but we are getting more genomic information about related crop species. So not just those that have been domesticated. It is going to have a significant impact on how we are going to develop the next crops, the next food, and some scientists are thinking of using genome editing also on microbials to moderate the composition of the microbiome. These tools are opening up new ways of conducting science, new types of products that could be developed because obviously, you can expand the genetic diversity to what is currently available.

Another informant, who is a research scientist working for an international organization, is excited about the applications of gene editing to microbes and how it can help reduce agrochemical use in food production. Informant 18 tells us,

[With] microbes, there’s a common phenomenon which is known as ‘quorum sensing’...Which is when one microbe communicates with another through sensing, to do the similar thing. It’s like a telephone or mobile phone or Twitter if you may…There is a process where nematodes are told through quorum sensing that there is no plant to infect. Can you beat that? So there’s a plant, there are nematodes, but they don’t go and infect this host plant because to them, there is no plant. So that means less or no pesticide spray. And nematodes are happy. The plant is happy. It’s feeding on something else, but not on your host plant.

There are remarkable innovations in the pipeline with gene editing that could revolutionize the agrifood system and make positive contributions to global food security. CRISPR-Cas9 is widely used to make these discoveries and make applications precise. However, there are other gene editing techniques on the horizon that use proteins other than Cas9 to edit the genome.

Open release products include plants, animals, microbes, and synthetic organisms that are designed to be released into the environment. The ability for gene edited organisms to exist without human intervention is cited as the major distinction between gene edited products in the past, and potentially, those in the future. The types of environments where these organisms will be released also varies. For example, some gene edited plants may be designed to exist in forests, pastures, or cityscapes, while microbes may exist underground in mines, waterways, in the soil, and in animal digestive tracts. Experts at the NASEM committee meeting thought that most of the products under development would be directed toward agrifood production, but also believed that they would be used for soil decontamination, and ‘lab’ meat derived from animal cells (NASEM, 2017).

Several novel open release applications of genomic techniques emerging from labs may create future regulatory challenges. Gene drive systems, often based on CRISPR-Cas9, allow an edited gene on a chromosome to copy itself onto its partner chromosome during cell division. Because the edited gene is copied on the partnered chromosomes, inheritance is 100% rather than 50%. ‘Cargo’ genes are required for a gene drive to work (Coffey, 2020). Cargo transgenes can be designed to confer chosen traits. Using CRISPR-Cas9 to introduce ‘cargo genes’ confers any trait that can be genetically linked to an engineered drive system. The genetic trait engineered via the drive can spread through a population. Gene drives alter an organism’s genes in a way that ensures that all offspring take on the edited traits, enabling a rapid spread of engineered genes through normal reproductive channels.

Scientists are experimenting with gene drives to control populations of invasive species or disease-carrying insects, such as the malaria-carrying mosquito of the Anopheles genus. Gene drives may also be gamechangers in terms of crop breeding (pest management, invasive species management). Research is under way to investigate potential applications to control agricultural weeds using these techniques (Neve, 2018). Over time, this could eliminate pest populations or decrease unwanted species in wild ecosystems (FAO, 2022). Researchers are working on developing gene drives that are limited to specific geographical regions that would help eradicate agricultural pests but are also adding immunization genes that could protect valuable and vulnerable populations of organisms (Bier, 2022).

Despite the promise of gene drives, there are concerns about unintentional consequences of using gene drives to genetically edit species for unconfined environmental release (Webber et al., 2015). Hayes et al. (2018) discusses potential hazards of open release gene drives into the environment. Risks include the introduction of gene drive organisms into non-target, related species in ecosystems. Hybridization or horizontal gene transfer could also have negative impacts on ecosystems. Nevertheless, to date there is little evidence of successful gene drive cases in plants.

Informant 12 (an academic researcher) spoke at-length about gene drives and why they are currently not a large part of the conversation on biotechnology in the agrifood system. They said,

there are a couple of different concerns. One is unrestricted spread. Usually, we want to control invasive pests and not native pests. And so native versus non-native is also remarkably important to ecologists. If you release a gene drive, that is a suppression drive that is really pushing down that pressure on replacement. That inoculates the insect, for example, from being able to host a certain pathogen in it. It could spread throughout native areas as well as the invasive areas. It’s very hard to control this kind of stuff. That’s how the invasive species got there in the first place. So technologically, are we there to control that spread? …there’s a trend towards more restricted and controlled use of gene drives.

While there remain questions about the ability to control gene drives once they have been released and their potential applications for benefits to agrifood production, there are other gene edited technologies in the pipeline that carry less uncertainty regarding unintended consequences.

3 Climate Change and Biodiversity

Gene editing offers potential solutions to help address agricultural challenges that have accompanied climate change. Plant domestication and genetic improvement have been important contributors to current levels of agricultural productivity and the global food supply. Improved crop yields ushered in by the first Green Revolution were achieved by creating high-yielding, lodging-resistant, fertilizer-responsive varieties (Vikram et al., 2015). The traits preferred during the domestication process for cereals for example, favoured a reduction in seed shattering, and absence of secondary dormancy,Footnote 2 while vegetable crops were bred for specific fruit size and shape, pigmentation, ease of planting/harvesting and transportation (Atwell et al., 2014; Fernie & Yan, 2019). However, many of the inherited traits involved in biotic and abiotic stress resistance may have been weakened or lost during this process of domestication for selected agronomic traits. While these domesticated crops perform well under ideal (temperature and soil moisture) conditions, these varieties are not necessarily suited to perform well under extreme climate conditions or on marginal soils. It has imposed limitations on the environments in which these crops can be efficiently cultivated. As climate change advances at a rapid pace, farmers are increasingly less able to rely on the ‘stable’ environmental conditions that these staple crops were bred to grow in.

Agriculture is extremely vulnerable to climate change. Many crops the world depends on were bred for temperature and precipitation ranges that are far less predictable today than previously, though farmers have always had to deal with extreme weather events such as droughts and floods, as well as pests. As Razzaq et al. (2021: 6124) state, climate change brings more frequent extreme weather events, which may lower long-term yields by damaging crops at various stages of development (Moriondo et al., 2010; Porter et al., 2014). As a result, the timing of field applications of fertilizers or pesticides is more difficult to predict, which can also contribute to decreasing yields (Antle et al., 2004; Tubiello et al., 2007). Changes in temperature, reductions in rainfall in certain areas and an over-abundance of precipitation elsewhere, influences the lifecycles of pests and negatively impacts soil composition. The quality of products will also be impaired, as elevated CO2 contributes to a reduction in protein content in cereal grains (Sinclair et al., 2000; Gornall et al., 2010). All these factors have put continued stress on the entire global agrifood supply chain, while contributing to growing global food insecurity.

The current progress in agronomy and crop breeding is not sufficient to keep up with the required increase in food production, prompting a need for a major shift in the breeding paradigm to create stress-resilient crops. The increasing efficiency and affordability of genome sequencing, and the development of novel genome editing tools opens new and exciting prospects for harnessing the potential of climate-resilient crop varieties, and investigating the genes present in wild relatives lost during the domestication process (Razzaq et al., 2021: 6133).

As such, researchers around the world are investigating how gene editing can help improve the resilience of ‘orphan crops’Footnote 3 that have long been neglected. So-called orphan crops are those which are not a major focus of crop breeding or international trade in agricultural commodities but may have a role to play in specific regions. There is also research underway regarding the potential of ‘re-wilding’ domesticated staple crops, like wheat and rice with traits from wild relatives that have been lost through domestication. But as climate change predictions point to warmer temperatures on the horizon, changes to rainfall patterns, and increased frequency and severity of extreme weather, the urgency to find ways of mitigating the effects of climate change on the world’s food supply have become of the utmost importance (Wheeler & von Braun, 2013; Zaid et al., 2020).

3.1 Orphan Crops

Orphan crops go by different names in the literature, including ‘underutilized’, ‘minor’, ‘neglected’, ‘promising’, ‘niche’ and/or ‘traditional’ (Yaqoob et al., 2023: 1). The category of orphan crops includes plants such as buckwheat, quinoa, cassava, banana, pigeon pea, millets, and many others. These crops are grown around the world by smallholder farmers in Latin America, Africa, and Asia. These crops are often resilient, can grow on marginal land, in lower quality soil and in some cases are more able to endure biotic and abiotic stresses than staple crops that have had some traits (beneficial to changing temperature and precipitation conditions) bred out of them to maximize yields in intensive monocropping. Some of the traits of orphaned crops make them less economical to farm on an intensive scale, such as smaller fruit size, low yield and less than ideal plant structure (Lyzenga et al., 2021: 142). We were curious to get an insight into how crops are chosen for genomic research, and eventually commercialization. In response to the question, ‘how are crops chosen for research purposes?’ Informant 17 (a research scientist) replied,

it’s acreage rate. And I’m not sure if ‘popularity’ is the word, but…There’s not a lot of external funding for [small acreage crops].

Because of the lack of their commercial importance, orphan crops have historically received less research funding and genomic resources than their staple counterparts.

Even though orphan crops have not been priority crops for research and investment, they hold a lot of potential to meet UN Sustainable Development Goals (e.g., zero hunger) in lower-income countries across Africa, Asia, and Latin America. Gene editing can be used to improve nutritional profiles of orphan crops and improve agrobiodiversity (FAO, 2022: 14). The FAO (2022) cites a number of organizations that are prioritizing genetic research into orphan crops for the public good, including CGIAR (formerly the Consultative Group on International Agricultural Research), as well as AIRCA centres (Association of International Research and Development Centers for Agriculture) such as ICBA (International Center for Biosaline Agriculture) and ICIPE (International Centre for Insect Physiology and Ecology), and NARS (National Agricultural Research Systems). The Gates Foundation is also conducting research into the potential of gene editing to enhance orphan crops like cassava (FAO, 2022: 14).

In response to the question, ‘How can gene editing be used to protect biodiversity and fight climate change? Are you a part of/are you aware of current research on gene-editing orphan crops?’, Informant 17 (a research scientist) responded:

…about the funding that rice, wheat or soybean would get, you would not expect it to be the same sustained effort that you would see for these millets or…legumes which are nutritious, more sustainable because I think they’re not put across that way or the focus is not there. But if you’re looking for something that is good for people, sustainable in the long run, we will have to find crops which fit that…and look for diverse crops, not just these three, four crops.

Wild relatives of orphaned crops may hold useful genetic traits that could improve agronomic outputs and nutritional profiles not found in cultivated crops. Informant 18, who works for an international development NGO, said that their organization’s ‘mandate crops’ are “chickpea, pigeon pea, ground nuts, sorghum and finger millet.” Some NGOs are researching how to use crops like pigeon pea to increase the productivity of marginal land. For example, Informant 18 is conducting research on what crops can be useful to intercropping strategies in India. As they explain,

we are also looking for plant architecture …for increasing yields in the case of pigeon pea especially. It should fit intercropping. What happens in many parts of India after they harvest rice, there is a very small window in which the field remains fallow. If that can be brought into cultivation with short duration pulses, you are taking a crop which would otherwise not be taken and providing diverse food on the plate as well.

Many other orphan crops offer potential. Table 4.1 below, adapted from Yaqoob et al. (2023), shows the type of crop and the trait(s) that may be useful as climate pressures challenge the outputs of higher profile crops like maize, wheat, rice, and soybean. Genomic research is underway to determine how to improve the agronomic profiles of these types of crops that have promise for global food insecurity.

Table 4.1 Major orphan crops and potential agronomic/food security characteristics
Full size table

Research is also underway to identify beneficial traits in wild relatives of orphan crops to improve agronomic profiles. De novo domestication has emerged as a potential way to harness the beneficial traits from wild species through gene editing and molecular breeding to create new, more climate resilient agrifoods. As Lyzenga (2021: 142) explains, many traditionally breeding techniques are ideal candidates for CRISPR-Cas gene editing platforms. However, challenges remain. There is a relatively high cost attached to phenotyping. The quality and control of seed systems and other crop inputs are other considerations in orphan crop research (FAO, 2022: 22). Without reliable seed distribution systems that farmers can depend on, the development of orphan crops, especially for smallholders in the developing world is much less impactful. The quality and control of seed systems and other inputs for crop production will play an important role in genomic research into orphan crops, as well as the pressing need to diversify agricultural production under the constantly changing environmental conditions of climate change.

3.2 Re-wilding

Re-wilding or ‘wide crossing’ involves taking traits from wild cousins of domesticated crops and using gene editing to improve genetic variation and biodiversity. Its goal is to reintroduce mutations into the cultivated crop gene pool that are still available in their wild relatives. The process of natural selection of wild relatives of some domesticated crops has resulted in an accumulation of genes that, for example, provide tolerance against pests, diseases, extreme temperatures, flooding, drought, and salinity (Montenegro et al., 2017). According to Cardi et al. (2023: 16), re-wilding can be done via introgression breeding (transferring genetic material from a species into the gene pool of another by backcrossing of a hybrid with one of the parent species), insertion of gene candidates and precision mutagenesis. CRISPR-Cas based systems can be used to knock out a gene, knock in a gene, and/or recombine genes at specific locations along the genome. It is something that is embarked upon not only by agricultural scientists, but environmental scientists looking for ways to mitigate the damaging effects of climate change.

The ability to use re-wilding as a strategy to combat climate change has been demonstrated in several plant varieties with the potential to become agrifood crops. For example, re-domestication of crop progenitors (wild relatives) in addition to the domestication of wild species has been demonstrated in Solanum pimpinellifolium (stress-tolerant wild tomato relative), Physalis pruinosa (groundcherry a distant relative of the tomato) and Oryza alta (wild tetraploid rice) by modifying domestication genes using CRISPR-Cas technologies (Cardi et al., 2023: 16). Other crops such as potato, kiwi, and pepper are the current focus of research into the ability to take genes from wild relatives to modify their modern, domesticated cousins. CRISPR has the potential to select appropriate genetic material to develop novel plant varieties combining good agronomic performance with adaptability to abiotic stresses and low input agricultural practices. The agrifoods listed in Table 4.1 are not typical staple agrifoods that populations depend on for survival, but research into re-wilding can yield important information about the potential to take traits from relatives of staple crops to make them more resilient to the effects of climate change, such as wheat.

Domesticated wheat has lost 70% of its genetic diversity compared with wild emmer (hulled wheat, a type of awned wheat) which had greater genetic diversity for abiotic and biotic stress tolerance (Haudry et al., 2007). Rice and soybean have lost 50% of their genetic diversity via domestication. Maize has lost 2–4% of its genes from the wild maize relative (Zea mays ssp. Paviglumis) through domestication processes (Razzaq et al., 2021: 6124). Some scientists argue that re-wilding could help combat the negative effects of climate change on agrifood production. It is a ‘tool in the toolbox’ that can be used when necessary.

Nevertheless, there are criticisms of the ‘re-wilding’ argument that recognize some of the challenges with this approach to improving resiliency in important staple crops. Informant 8 (a private sector representative) expressed some skepticism in terms of the viability of what they called ‘wide-crossed’ crops. As they said,

it still tends to be a last resort for plant breeders, and it would generally only be done when they’ve exhausted other options.

There is significant upstream work that would be needed to find desirable traits in wild relatives, and years of research that would be necessary to find appropriate genes to knock out, knock in, or recombine. Wild varieties may not yet have their genomes sequenced, which would be the first step to investigating what traits could be re-introduced into their domesticated relatives. The very first step to realizing the potential of orphan crops and re-wilding important agrifood crops would be to sequence the relevant genome, and experiment with various gene edits to develop promising plants that can adapt to future climate uncertainties.

4 Beyond CRISPR-Cas9

Several emergent platforms do not rely on CRISPR-Cas9 for gene editing. As Labant reports in 2022’s Genetic Editing and Biotechnology News, a new system for gene editing known as ARCUS has been discovered by scientists. This consists of enzymes derived from I-CreI, a shellfish genetic element that occurs in the algae Chlamydomonas reinhardtii. Other starting materials from nature include mobile genetic elements (MGEs), which have potential as “gene writing” tools that eschew double-strand breaks (Labant, 2022).

Other gene editing techniques do not rely on the Cas9 protein to manipulate the genome. MAD7 for example, is gaining popularity. MAD7 is a CRISPR enzyme that is similar to Cas9 and Cas12a.Footnote 4 Class II type V CRISPR-Cas12a is a new RNA guided endonuclease that has been recently harnessed as an alternative genome editing tool, which is emerging as a powerful molecular scissor to consider in the genome editing application landscape (Bijoya & Montoya, 2020). MAD7 is freely available from the patent holding company (Inscripta) and has been made available for use in research and commercial R&D so that it can be tested (Inscripta, 2021).

In 2021, the first successful use of MAD7 in a plant genome was discovered by a team of scientists at the Chinese Academy of Sciences in Beijing (Lin et al., 2021). An article in the Journal of Genetics and Genomics describes how the team use the MAD7 nuclease to genetically alter rice and wheat, demonstrating its potential for engineering crops. The results demonstrate the editing efficiency of MAD7 in rice and wheat. It is up to 65.6% successful in producing edits, comparable to that of the widely used LbCas12a CRISPR system. Despite being one of the most robust Cas12a nucleases, LbCas12a in general is less efficient than SpCas9 (another naturally present nuclease) for genome editing in human cells, animals, and plants. Cas12a nucleases, is thus called ‘LbCas12a’. Additionally, the authors demonstrate that this approach can be used for multiplex gene editing when used with other CRISPR orthologues.Footnote 5 In the second publication in Progress in Molecular Biology and Translational Science, the authors discuss the MAD7 nuclease as an important resource that can overcome current limitations of CRISPR editing:

A wide range of families and orthologues of CRISPR-associated proteins are being developed to fill the gaps in genome engineering by increasing their functionality, specificity, and […] and ease of access globally (Bayarsaikhan et al., 2021).

MAD7 was a topic of discussion for many of the research scientists we interviewed. Some seemed excited about the prospect of a CRISPR-based gene editing system that is not based on a royalty payment model, as MAD7 is free to use and develop gene edited products with commercial potential. As Informant 17 said,

There are some limitations to MAD7 as far as it is based on its some of the biochemical properties…my lab’s exploring that as well. Because of the unique licensing approach…academic labs, government labs would be able to develop traits that would allow small and medium companies to come up and run with them…So it’s an important area of research but, CRISPR-Cas9 is the gold standard in terms of precision and accuracy.

Informant 5 was intrigued by alternatives to the Cas9 system, but pointed out that,

there was path dependence, because the regulators, once they’d approved it 3 or 4 times, said ‘we’re good with that, we understand there’s no risk with that promoter using this technology’. But every time you bring in a new promoter you got to go back to square one and prove the basic safety. You can invent a new one but it may not make any economic sense, because unless you can find one that you get enough volume out of that the people will invest in, taking to market will be just too expensive to reinvent. So it’s cheaper to license from the other one.

Some platforms may be able to get better results, or different results, but may face regulatory hurdles if regulators are not familiar with the mechanisms of change.

The potential of CRISPR-Cas systems to be applied to transgenic crops is also being explored. Informant 12 (an academic researcher/social scientist) sees the future of gene editing as a useful application to transgenic crops to achieve multiple desired traits. They say,

…a lot of gene editing is probably going to happen, like further transforming products that are already transgenic. There are a couple of traits that are extraordinarily valuable that you cannot get. They are from foreign DNA, for example, Bt. It is an extraordinarily valuable innovation and is from a bacteria. That’s the whole point…maybe we’ll find some equivalent of proteins or something. But that’s not going away anytime soon, I don’t think whatsoever. And there’s herbicide tolerance. This is achieved through transgenic methods…My understanding is that there is not a knockout strategy for herbicide tolerance, and herbicide tolerance is also an extraordinarily economically valuable asset.

We briefly discuss three other approaches to altering the genomes of agrifood: base editing, prime editing, and RNAi technology. Base editing is an evolution of CRISPR-Cas systems. It does not require a Double Strand Break (DSB), which many proceeding gene editing technologies require. Base editing has emerged as an alternative tool to homology-directed repair (HDR) mediated replacement. Since there is low efficacy of HDR in plants, base editing can allow for precise nucleotide changes in the genome. Base editing facilitates precise editing of a plant genome by converting one single base to another in a programmable manner. For single-base substitution, base editing is emerging as an alternative and efficient powerful tool to HDR-mediated precise gene editing in plants. Base editing can be used to improve yields, nutritional qualities, pest resistance, and herbicide resistance in plants. Since base editing does not require DSBs or donor templates, they are a new way to think about ways to edit plant genomes in agriculture (see Li et al., 2023).

Like base editing, prime editing can make changes to a DNA strand without the DSBs. The prime editing system is derived from the CRISPR-Cas system. Prime editors, unlike other techniques, do not require DSBs or a donor DNA template to make gene edits. They require a programmable ‘cutting enzyme’ (nickase) such as the Cas9 protein, that is capable of cutting the DNA at a specified site. The nickase is fused to a polymerase enzyme which is an enzyme that synthesizes DNA or RNA and assembles DNA or RNA molecules. They can make almost any substitution, deletion, or insertion in the DNA of living cells. The feasibility of using Plant Prime Editing (PPE) in crops was demonstrated in 2019, but its efficacy was considered too low, especially compared with CRISPR-Cas systems. There is, however, significant current research into how to improve PPE’s efficacy so it can be used effectively in plant breeding. Its efficacy has been demonstrated in herbicide resistance germplasm. There are current limitations to this approach, such as the inability for it to insert large sequences. There are also limitations of PAM sequences (Protospacer Adjacent Motif is a 2–6 base pair DNA sequence following the sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system). Informant 16 (a research scientist) commented that

…[there are] only a handful of labs in the world that actually tried prime editing. Prime editing might allow us to introduce very specific changes wherever we want them.

PPE has a lot of potential for precision editing and synthetic biology in agricultural plants, when research has revealed how to make it as efficient at editing for the desired traits as its counterparts (Tingting et al., 2023).

Finally, we turn to RNAi technology, which is an older technology relative to gene editing per se. The primary function of natural RNA interference is to regulate gene expression. RNA interference (RNAi) is a natural process found in most eukaryotic organisms (plants, animals, fungi) that was first identified in the late 1990s and was found to supress gene expression in a sequential order. Andrew Fire and Craig Mello won the 2006 Nobel prize for Physiology or Medicine for their ground-breaking work on how double strand breaks were responsible for gene silencing. Compared to CRISPR, RNAi technology reduces gene expression at the RNA level, what is known as ‘knock-down’, while CRISPR permanently silences the gene expression at the DNA level, what is called a ‘knock-out’. Gene knock-down is important for research purposes because researchers can see the effect on a phenotype since reduced protein levels can be measured. Knock-downs are also reversable, so a researcher can return the protein expression to normal to observe the changes that the knock-down induced (Mezzetti et al., 2020).

One of the major downsides to using RNAi is off-target effects. CRISPR-Cas systems are much more precise and have less instances of off-target effects. Despite these challenges, researchers are experimenting with RNAi techniques to improve upon agronomic traits of plants such as targeting pest and pathogen genes within the plant’s genome, as well as looking at surface applications. Decades of research using this technique has revealed target genes that can improve tolerance to biotic and abiotic stresses. RNAi technologies also have the capacity to down-regulate gene expressions without disrupting the expression of other genes. Though RNAi technology is much older than CRISPR-Cas systems it is another available tool in the toolbox for research scientists to help solve some of the most pressing challenges facing agriculture and food security.

5 Conclusions

The world of gene editing in agrifood is constantly changing. New discoveries are made everyday with the potential to revolutionize how we produce food and how the world feeds itself. In this chapter, we have discussed some of the newest breeding techniques being used in research labs around the world. It is important for regulatory systems to prepare for the wave of new breeding techniques on the horizon. Chapter 5 discusses regulatory change and the regulation of futures technologies.