Precision breeding or gene editing is increasingly recognised as a critical tool for improving global food security and sustainability, with over 500 precision-bred crop products in development worldwide. Gene editing enables the implementation of complex traits into agricultural products, without the need for long breeding cycles. Importantly, this approach is the primary method used to introduce novel traits into sterile crops such as banana. Significant strides have been made in implementing precision breeding (e.g., using CRISPR/Cas9 technologies), while commercial adoption is increasingly enabled by favourable regulations, exemplified by the entry into force in England of the Genetic Technology (Precision Breeding) Act 2023, which permits the development and marketing of precision-bred crops and livestock. Yet, precision breeding continues to face a significant bottleneck at the gene discovery stage. While traditional breeding screens have been vital to identify trait genes, such a technique can take many decades and cannot be employed in sterile crops such as banana. Alternative gene discovery methods are required to enable trait development in crops that have long-breeding cycles or are sterile. Innovate UK funding brings together a world-class industry-academic consortium, entirely based in Eastern England, to address the gene discovery bottleneck and unlock precision breeding. Project lead Tropic (global leader in advanced plant breeding and gene editing technologies, based in Norwich) will collaborate with the John Innes Centre (JIC; a world-leading international centre of excellence in plant science, genetics, and microbiology, based in Norwich), and AI Digital (service delivery consultancy based in Lincoln, with expertise in life science applications). Our integrated experimental and bioinformatics approach will accelerate novel trait discovery for precision breeding by addressing challenges in the generation, analysis and visualisation of large-scale multi-dimensional functional datasets. We will develop and demonstrate our innovative approach in banana, the world's fourth largest crop by production volume. Banana plays a vital role in global food security and ensuring the livelihoods of millions of small-scale farmers. Yet, banana production is increasingly threatened by climate change, with rising global temperatures and more extreme weather conditions reducing yields, as well as driving a rise in rapidly spreading emerging pathogens. Tropical race 4 and black-sigatoka disease are already causing global annual banana production losses of billions of dollars. Bananas also suffer from high wastage throughout their supply chain. Over 60% of exported bananas go to waste in the supply chain (i.e. before reaching the consumer), often because of browning and bruising.

Over 185 million metric tonnes/year of tomatoes are produced globally worldwide on 5.1m hectares (Faostat, 2020), with a global market value of nearly USD200bn (Mordor Intelligence, 2023). Yet, global tomato production faces an unprecedented threat from an emerging viral pathogen, tomato brown rugose fruit virus (ToBRFV), a seed- and contact-transmitted virus that causes high infection rates (up to 100%) and severe crop yield losses (ranging from 25 to 70%). ToBRFV particularly affects greenhouse tomato production because of the worldwide seed distribution for greenhouse tomatoes and the increased need for plant handling compared to in open-field production, increasing the risk of mechanical transmission. The first outbreak of ToBRFV was in Israel in 2014 and all tomato production areas in Israel remain severely affected by ToBRFV, with national yield losses of approximately 50%, responsible for estimated economic losses of £26m/year (Fresh Produce Journal, 2021). Today, the virus has been reported in at least 35 countries worldwide, with the first outbreak in the UK reported in 2019\. The combination of virus pressure and high energy prices continues to threaten commercial viability for greenhouse tomato growers. In the UK and elsewhere, winter production during 2022/23 was significantly reduced. Spring 2023 saw tomato shortages in the UK, with UK tomato prices increasing by 22.3% compared to the previous year (ONS, 2023). Through this UK-Israel Eureka Bilateral Collaborative R&D: Round 2 project, UK-based Tropic and Israel-based BetterSeeds will combine their complementary skills in plant genome editing to develop durable and complete resistance to ToBRFV, which can be scaled across high-performing tomato varieties without incurring a yield penalty or reducing product quality. Founded in 2016, Tropic is a pioneering agricultural-biotechnology company developing advanced plant breeding and gene editing technologies, employing over 140 industry-leading professionals. BetterSeeds is Israel's largest and leading plant genome editing company, founded in 2017 and currently employing 25 people, predominantly researchers in plant science and genetics. Ultimately, successful project outcomes will protect greenhouse tomato growers from catastrophic yield losses resulting from ToBRFV outbreaks, reducing unproductive operational costs, improving productivity and profitability, while ensuring a resilient supply of healthy and sustainable fresh produce to consumers.

Facing the triple challenge of feeding a growing population, dealing with the impacts of climate change, and preventing major losses to emerging pathogens, gene-editing technologies are increasingly seen as playing a key role in achieving future global food security. Unlike genetically modified organisms (GMO), which contain genetic changes that could not have occurred through traditional breeding or arisen naturally, regional regulations are increasingly permissive for gene editing, which focuses on genetic changes that could have arisen through traditional breeding methods or occurred naturally. The UK is a global leader in genetics and genomics research and this position has recently been underpinned by the entry into force of world-leading gene-editing regulations through the Genetic Technology (Precision Breeding) Act 2023, which permits the development and marketing of gene-edited crops and livestock in England. Founded in 2016, Tropic is a pioneering agricultural-biotechnology company developing advanced plant breeding and gene editing technologies, employing over 140 industry-leading professionals at our headquarters in the Norwich Research Park Innovation Centre. We are developing high-performing commercial varieties of tropical crops in-house (focusing initially on banana, rice, and coffee) and, outside tropical crops, making our proprietary and patented gene-editing technology platform available to select partners including leading global agribusinesses BASF, Corteva Agriscience, and Genus. Banana is the world's fourth largest crop by production volume, playing a vital role in global food security and ensuring the livelihoods of millions of small-scale farmers. We are developing gene-edited bananas with valuable traits including extended shelf-life and non-browning, as well as resistance to tropical race 4 (TR4) and black-sigatoka disease (BSD). Banana browning is responsible for the loss of over 60% of exported bananas in the supply chain (i.e. before reaching the consumer). TR4 and BSD are rapidly spreading fungal pathogens that threaten global banana production, with BSD already causing global annual banana production losses of £1.1 billion and BSD management accounting for 27% of banana production costs. Since cultivated bananas are triploid and sterile, new varieties cannot be produced through conventional breeding. While gene editing offers significant promise for valuable trait development, gene editing of banana faces significant implementation challenges including low transformation efficiencies and the requirement to develop non-transgenic transformation methods (since transgenes cannot be bred out). With Innovate UK funding, we will develop efficient and non-transgenic transformation methods for sterile and/or recalcitrant crops, focusing initially on banana as a model crop system.

The UK is approximately 50% self-sufficient in sugar production, with 2,300 English sugar beet farmers based in East Anglia and the East Midlands producing 7.4 million tonnes of sugar beet/year, valued at £216m (2021). Yet, the English sugar beet industry faces an existential threat from virus yellows, which in 2020 infected 38.1% of the English sugar beet crop, costing English sugar beet farmers approximately £43m. Virus yellows is transmitted by aphids (principally peach potato aphids) and consists of three viral species; namely, Beet Mild Yellowing Virus (BMYV), Beet Chlorosis Virus (BChV), and Beet Yellows Virus (BYV). BYV is the most damaging of the yellowing viruses, causing yield losses of up to 50%, and was also the prevalent viral species detected in English sugar beet in 2020\. With climate change driving warmer, wetter winters in England, infective aphids are increasingly likely to successfully overwinter and migrate earlier, seriously impacting emerging sugar beet seedlings, as exemplified in England's 2020 virus yellows outbreak. Today, English sugar beet farmers have limited options available, since previously widely used and relatively effective neonicotinoid pesticide seed treatments have been banned in the UK and Europe since 2018 because of growing evidence of the severe impact these pesticides have on the survival of bees and other pollinators. However, with sugar beet growers facing serious economic losses and with no alternative options available, the UK has, for the last three years, granted Emergency Authorisations permitting the use of neonicotinoid pesticide seed treatments where conditions require them and subject to strict controls and measures. The industry and regulators are keen to move away from neonicotinoids and last year, the EU's high court banned emergency exemptions. The English sugar beet industry is in urgent need of a long-term, sustainable solution to tackle the existential threat of virus yellows. Through the Farming Future R&D fund, British Sugar (world-class sugar producer and the sole processor of the UK's beet sugar crop), Tropic (pioneering agricultural-biotechnology company developing advanced plant breeding and gene editing technologies), and the John Innes Centre (international centre of excellence in plant science, genetics, and microbiology) will further develop a precision breeding pathway via gene editing to confer virus yellows resistance in sugar beet.Ultimately, successful project outcomes will protect English sugar beet farmers from potentially catastrophic losses to virus yellows, increasing productivity, resilience, and sustainability, while supporting progression towards net zero emissions in English agriculture.

**Challenge:**Rice is a staple for nearly half of the world's 7 billion population and is mainly produced in south-east Asia (Mohanty\_2013). As the global population rises, to over nine billion by 2030, there will be huge pressure on land for food -- and rice yields must increase by 25% (Fernandez\_and\_Orth\_2018). However, rice yields are decreasing due to climate change (Zhao\_et\_al\_2017). In addition, rice cultivation accounts for 12% of global methane emissions (World\_Economics\_Forum\_2019), which is 30-times more a potent Green House Gas, than CO2 (National\_Geographic). Today, global rice production is doing as much harm as 1,200 average-sized coal power stations, and by 2030, will be responsible for 6% of total GHG emissions (We\_Forum\_2019). Despite sustained initiatives particularly in China, Japan, Korea and at the International Rice Research institute (IRRI) in the Philippines, established approaches are unable to produce high enough yields, and have effectively plateaued in China, Indonesia, Japan and Korea, and although rising linearly in some key countries such as India and Vietnam, the rates of increase are too slow to meet demand (Grassini\_and\_Cassman\_2013). **Solution:** This 24-month industrial research project between NIAB and Tropic advances the state-of-the-art in a plant product (rice) and it's production system (gene editing to increase rice yield), creating a unique UK technology and product export opportunity, and significantly contributes to Net Zero (reducing emissions from rice cultivation), demonstrated through glasshouse trials initially with field performance confirmation latterly (TRL7). We have already demonstrated that we can gene edit favourable traits into a variety of crops (e.g., disease resistant bananas and rice). We will use our previous know-how to build this novel production system, including technical work, utilising Knock-Down and Knock-Out gene editing to modify yield-related regions of interest in rice and select the best rice lines. By the end of this project, we will be in a position then utilise our patented 'GEiGS' technology to create non-GMO rice in several rice varieties, which we will then commercialise. Impact: Increasing rice yield, has the potential to massively cut GHG emissions, whilst producing more grain, and thus keeping the land-mass utilised constant. We have predicted that higher yielding rice could lead to a total CO2 reduction for rain-fed rice of 730 Kg CO2/ha, and for irrigated rice 1330 Kg CO2/ha. **_If everyone globally were to consume Tropic's rice, this could equate to savings of 433 million tonnes of CO2 annually_.**

By production volume, bananas are the fourth most important global crop, with 115 million tonnes of banana produced in 2019 (FAO, 2021). Most banana cultivation is performed by small-scale farmers, providing a vital source of income and employment to over 70 million people in Africa alone (IISD, 2020). However, global banana production is increasingly threatened by the rapid spread of Black Sigatoka Disease (BSD), also known as black leaf streak. BSD causes leaf necrosis and can decimate local crop yields, with a catastrophic impact on smallholder livelihoods. BSD is already responsible for reducing global annual banana production by an average of 3%, equivalent to an annual loss of around £1.1billion (Strobl and Mohan, 2020). Since cultivated bananas are triploid and sterile, new BSD-resistant varieties cannot be produced through conventional breeding. Instead, farmers are reliant on managing BSD through frequent (weekly) fungicide treatments applied throughout the growing season (Bellaire et al., 2010). With a fungicide treatment cost of up to £1,000/ha, this is extremely expensive for large plantations and prohibitively expensive for small-scale farmers (Onyilo et al., 2018). Fungicide application is also driving increased pathogen resistance. Globally, BSD management already accounts for 27% of banana production costs, concurrently increasing the global banana price (Yonow et al., 2019). Climate change (increasing rainfall and temperatures in banana-growing regions) is predicted to further increase the spread of BSD (Bebber, 2019). Tropic Biosciences is developing high-performing commercial varieties of tropical crops including banana, coffee, and rice, which promote cultivation efficiencies, enhance consumer health, and improve sustainable environmental practices. Our approach relies on proprietary, patent-pending cutting-edge gene editing (GE) technologies, developed in house. With Innovate UK support, we will combine our world-leading GE technologies with a novel breakthrough GE technology, developed by the Broad Institute of MIT and Harvard. As a demonstration of the potential of our new game-changing biotechnology, we will develop BSD-resistant banana plants, reaching laboratory validation stage. This project's outputs have the potential to play a key role in supporting the UK's ambition to become a world leader in Bio-/Agri-Tech, in close alignment with the goals of the ISCF Transforming Food Production and addressing five UN Sustainable Development Goals: 1 (No Poverty), 2 (Zero Hunger), 5 (Gender equality), 12 (Responsible consumption and production), and 15 (Life on land).

Bacterial diseases of plants and animals cause devastating damage to crops and livestock, respectively, resulting in significant global economic losses (worth multiple billions of pounds annually). With the rise of antibiotic-resistant bacteria threatening human health, agribusinesses are under pressure to reduce antibiotic use. Development of antibacterial resistance through gene editing(GE) shows promise, but remains limited by slow/costly gene discovery. Bacteria are one of the major pathogens causing emerging infectious plant diseases. One high-profile example is citrus greening or huanglongbing, which is recognised as the most devastating citrus disease worldwide(Dala-Paula\_et\_al\_2019). Citrus greening affects tree health as well as fruit development, ripening, and quality. There is currently no cure or management strategy, with infected trees dying within as little as 5years(Singerman\_and\_Rogers\_2020). In the US, over the last decade, citrus greening has caused Florida's orange production to decrease by over 70%(USDA\_2019), and the price of a box of oranges to increase by over 3x(Dala-Paula\_et\_al\_2019). Tropic Biosciences is developing high-performing commercial varieties of tropical crops (including banana, coffee, rice), which promote cultivation efficiencies, enhance consumer health, and improve sustainable environmental practices. Our approach relies on proprietary/patented cutting-edge GE technologies and has overcome the gene discovery barrier. However, our current technical approach is limited to targeting plant/viral/fungus/pest genes and cannot target bacteria. Headquartered at renowned Norwich Research Park, with access to world-leading facilities, Tropic Biosciences was founded in 2016 by a team of highly experienced entrepreneurs and academics, including: CEO Gilad Gershon, experienced AgTech private equity investor/entrepreneur; CSO Dr Eyal Maori, scientific co-founder of Beelogics, acquired by Monsanto(2011); and, CTO Dr Ofir Meir, former Head of the Agro Chemistry Division at Rosetta Green, acquired by Monsanto(2013). We now employ a team of 80+ industry-leading experts and have raised £22.5million Series B round (largest ever in the UK AgriTech sector) to accelerate our growth trajectory through field trials for our novel banana varieties and to further commercialise our proprietary Genome Editing induced Gene Silencing(GEiGS) technology platform. While GEiGS enables host protection against viruses, fungi, and pests, it cannot target bacteria. This R&D project extends our technical approach to bacteria for the first time and is highly complementary to GEiGS. A successful project, validating experimental proof of concept(technology readiness level 3) will unlock in-house development of high-performance antibacterial tropical crop varieties, resistant to emerging bacterial plant diseases. Through licensing to established agribusinesses, Hi-GE platform will also enable development of antibacterial traits in non-tropical crops and animals.

Rice is a staple food source for over half the world's population and the main source of income and employment for over 200 million households across the world (Asibi\_et\_al\_2019). World rice production is forecast to reach over 500 million tonnes in 2020 (FAO\_2020). However, rice production cannot meet increasing global demand, with the world population projected to reach 8.5 billion in 2030 (UN\_2019). Rice yields are also decreasing as a result of climate change and rising temperatures; each 1°C rise in global mean temperature is predicted to reduce the global rice yield by 3.2% (Zhao\_et\_al\_2017). Today, 30% of rice production losses globally (which could feed 60 million people; Nalley\_et\_al\_2016) are caused by rice blast, a pathogenic fungus that can cause total crop failure and is found in all rice-growing regions (Wang\_et\_al\_2014). The appearance of new and more virulent strains of rice blast are challenging for traditional management and control methods such as fungicides and plant breeding (Asibi\_et\_al\_2019). Tropic Biosciences is developing high-performing commercial varieties of tropical crops (such as banana and coffee), which promote cultivation efficiencies, enhance consumer health, and improve sustainable environmental practices. Our approach relies on proprietary and patented cutting-edge genetic editing technologies, developed in house. With Innovate UK support, we will extend our world-leading gene editing technology to the development of rice blast resistance, building our portfolio in rice. Headquartered at the renowned Norwich Research Park, with access to world-leading facilities, Tropic Biosciences was founded in 2016 by a team of highly experienced entrepreneurs and academics, including: CEO Gilad Gershon, experienced AgTech private equity investor and entrepreneur; CSO Dr Eyal Maori, scientific co-founder of Beelogics, which was acquired by Monsanto in 2011; and, CTO Dr Ofir Meir, former Head of the Agro Chemistry Division at Rosetta Green, acquired by Monsanto in 2013\. We now employ a team of over 80 industry-leading experts. This year, we closed a £22.5 million Series B round of equity funding (the largest ever funding round in the UK AgriTech sector) to accelerate our growth trajectory through field trials for our novel banana varieties and to further commercialise our proprietary Genome Editing induced Gene Silencing (GEiGS) technology platform. This Innovate UK project extends our work to rice for the first time. By achieving a sustainable increase in rice production, this project will improve global food security and contribute to poverty alleviation, in the face of global population growth and climate change.

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Black Sigatoka disease (BSD) is an airborne fungal disease which cripples E. African banana crop production. Typical management of BSD relies on frequent (weekly) fungicide treatments throughout the growing season. However, in E. African the use of fungicides is beyond the reach of many resource-constrained subsistence farmers, and when they are used, fungicides present significant environmental and health hazards. Improving disease resistance through high performance crops is extremely challenging in the banana industry. New banana varieties cannot be produced through conventional cross-breeding as Cavendish bananas are triploid and sterile. In partnership with the IITA we will develop and demonstrate, 2 new non-transgenic BSD resistant varieties of banana (Grand Naine, Dwarf Cavendish) in a relevant economic and environmental setting. Using our proprietary GEiGS and NTC systems we will create programmable resistance to BSD utilizing RNAi within the plant cell, enabling E. African growers (notably in Uganda, Kenya, Tanzania) increased productivity (10-25%) by enabling closer spacing of individual plants and reduced harvest loss (BSD can decimate upto 60% of harvest weight).

"GMO crops incorporate foreign DNA into the genome to create specific improvements such as yield increases or disease tolerance. However, this methodology requires extensive development processes, strict regulatory approval pathways and encounters strong public stigmatization. There is a new method for crop protection which utilizes RNA interference (RNAi), which promises to allow us to address some of agriculture's greatest priority challenges (including root knot nematodes \[$10'sBn damage annually\], _Potato leafroll polerovirus_ \[$100'sM damage annually\]) and increase food security on a global scale. However, there is a significant technological barrier which is preventing RNAi being used in most crops in a non-GMO manner. We are developing a unique and highly innovative approach to enable RNAi mediated disease protection to be imbued to all agricultural crops in a non transgenic manner."

"GMO crops incorporate foreign DNA into the genome to create specific improvements such as yield increases or disease tolerance. However, this methodology require extensive development processes, strict regulatory approval pathways and strong public stigmatization. There is a new toolkit (CRISPR-Cas9) which has revolutionised non-GM breeding since its adoption in 2012\. It allows for low cost, highly targeted edits of cells, without incorporating foreign DNA. In fact, the mutations it creates could arise naturally, it just allows for focused efforts and removes random chance. However, there is a significant technological barrier which is preventing CRISPR-Cas9 from being employed in many crops. This is what Agro-Trop-RNP addresses. We are developing a unique and highly innovative approach to enable the whole CRISPR-Cas9 toolkit to be applied to all agricultural crops. This will allow us to address some of agriculture's greatest remaining challenges (eg. Black Sigatoka Disease in bananas causes 50% yield losses, obliging fungicide application 20-70 times/yr: increasing production costs by 25%) and increase food security on a global scale."

Crop productivity must increase 70% by 2050 to meet rising global population (WHO, 2016). Genome Editing (GE) increases productivity by creating new plant traits. Payments to use the technology for a single species range from £8m-80m (AgFunder, 2016). I is a complex 2-stage generation process, with the 2nd stage (non-transgenic GE) especially lengthy. All crops are at risk of disease, eg Black Sigatoka Disease (BSD) causes 50% yield losses in banana, obliging farmers to use fungicide 20-70 times/yr (Fairtrade, 2016). Thus, to make non-transgenic GE available to more species, such as asexual crops and crops with long breeding cycles the process needs to be faster and cheaper (Economist, 2016). Tropic Biosciences (TBio) has created a Non-Transgenic GE (NTC) Tool, a single-stage only generation GE process, meaning it can be applied to long breeding cycle and asexual crops. Their objective is to prove the concept in lab conditions, demonstrate it with a BSD-resistant banana trait, reduce the GE process time and cost by 70% and make their NTC Tool affordably licensable to small innovators.