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By Jun Guo I'm excited to share some groundbreaking research that's been making waves in the world of plant genetic engineering. A team of researchers from diverse fields unveiled the innovative Cut-Dip-Budding (CDB) CRISPR-cas9 construct delivery system, which promises to revolutionize how we modify plants genetically. This method has been published recently in the scientific journal, Innovation and Plant Biotechnology Journal, directed by Dr. Jian-kang Zhu at the Southern University of Science and Technology, China. It is poised to revolutionize research and breeding practices worldwide! Does tissue culture have to be involved when we do gene modifications? CRISPR stands for Clustered Regularly Interspaced Short Palindromic Sequences that together with the enzyme Cas9 (CRISPR Associated) is used as a precise gene editing tool in plants. Traditionally, genetic modification technology in plants using CRISPR-cas9 has heavily relied on tissue culture techniques using Agrobacterium tumefaciens, which can be both complex and costly. Tissue culture is a sterile technique where scientists provide plant stem cells or 'explants' with hormones that can regenerate a full plant. However, the CDB delivery system offers a transformative alternative. Rather than relying on labor-intensive tissue culture, this method involves directly inoculating plant explants with the related root transforming bacterium - Agrobacterium rhizogenes. Here's how it works: The Cut-Dip-Budding delivery system employs Agrobacterium rhizogenes to deliver the CRISPR-cas9 construct, enabling target gene editing in regenerated roots or shoots. Agrobacterium rhizogenes triggers the formation of transformed roots in plant explants. Subsequently, these transformed roots give rise to transformed buds through a process known as ‘root suckering’ whereby the plant responds to wounding by making adventitious buds that give rise to clonal plants. What's truly remarkable is that this entire process occurs without the need for the elaborate setups and sterile conditions typically associated with tissue culture (Figure 1). In the study by Zhu, the findings suggest that succulents with shoot regeneration ability from cut leaves can be genetically transformed using the CDB method, thus opening up an avenue for genetic engineering of these plants. Figure 1: Succulents with shoot regeneration ability from cut leaves using the CDB method. Why is the CDB delivery system revolutionary? The beauty of the CDB method lies in its simplicity. Rather than navigating through complex protocols, researchers can use a straightforward explant dipping protocol that can be carried out under non-sterile conditions, making the process more accessible and cost-effective. By simplifying the process of genetic modification, this innovative approach accelerates the pace of crop improvement, facilitating the development of resilient, high-yielding varieties tailored to diverse agroecological conditions. What's particularly exciting is the broad applicability of the CDB method. Researchers have successfully demonstrated its effectiveness across various plant families, including species that were previously challenging to modify genetically. The CDB delivery system helps bypass the major bottleneck in CRISPR applications – gene delivery in non-model plant species. Final thoughts: The Cut-Dip-Budding Delivery System represents a monumental leap forward in plant genetic engineering, characterized by its simplicity, accessibility, and effectiveness. As we embrace this new era of precision breeding, the future of crop improvement looks brighter than ever before. In my own research on Aluminum stress responsive proteins in tomato roots, I find some genes only or mainly express in the roots. If this method works for my system, maybe it can be used to functionally validate several genes likely involved in aluminum tolerance. References Cao, X., Xie, H., Song, M., Lu, J., Ma, P., Huang, B., ... & Zhu, J. K. (2023). Cut–dip–budding delivery system enables genetic modifications in plants without tissue culture. The Innovation, 4(1). Lu, J., Li, S., Deng, S., Wang, M., Wu, Y., Li, M., ... & Zhu, J. K. (2024). A method of genetic transformation and gene editing of succulents without tissue culture. Plant Biotechnology Journal. Lu, J., Lu, S., Su, C., Deng, S., Wang, M., Tang, H., ... & Zhu, J. K. (2024). Tissue culture-free transformation of traditional Chinese medicinal plants with root suckering capability. Horticulture Research, 11(2), uhad290. Acknowledgement This article was written with AI assistance for language editing. Jun Guo is a final year PhD student at Tennessee State University, specializing in Agricultural Sciences and Engineering. His research is dedicated to developing tomato Aluminum stress-tolerant lines using CRISPR technology. Beyond academia, Jun finds solace in nature through running and hiking.

By Kyla Hughes In a perfect world, humans would be free of diseases, illnesses, syndromes, and other conditions. Unfortunately, many people worldwide face some form of illness varying from manageable to debilitating (GBDS Collaborators, 2015). One specific, prevalent condition is Immunoglobulin E (IgE)-meditated food allergy, commonly referred to as “food allergies.” IgE food allergies have increased in prevalence in the United States and across the world in recent years. Approximately 1 in 10 adults and 1 in 12 children in the United States suffer from food allergies from at least one of eight major food allergens: soy, wheat, dairy, eggs, fish, shellfish, peanuts, and tree nuts (Gupta et al., 2019). For people who have food allergies, avoiding certain foods can be troublesome. In some cases, even after avoiding them, an accidental interaction with a specific allergen can lead to illness, hospitalization, or even death (Warren et al., 2020). Having a food allergy can also be a financial burden; from annual visits with allergists to buying allergen-free food products to purchasing expensive, prescribed Epinephrine. For these reasons, and many more, allergists and scientists are conducting research to identify if the burden of IgE food allergies can be relieved for those who suffer from them day-to-day. Image Source: Flickr USDA gov https://www.flickr.com/photos/usdagov/27239936717 One important question scientists have begun to ask themselves is: Can food allergens be genetically removed from major food crops using CRISPR engineering? Certainly! In recent decades, CRISPR (Clustered Regularly Interspaced Short Palindromic Sequences) editing has shown promising results in agricultural, medical, and other related fields. This past year, the Unite States Food and Drug Administration approved the first gene therapy to treat sickle cell disease (SCD); this therapy utilizes CRISPR technology (FDA, 2023). In the agriculture sector, CRISPR has the potential to improve crop yields, reduce the need for pesticides, herbicides, and fertilizers, and promote plant growth in the face of climate change. On the molecular level, CRISPR can be complex, however, learning the fundamentals will certainly be useful even for non-scientists. When discussing IgE food allergies, CRISPR serves as a precise engineering tool to completely delete genes relating to allergy. Using a Cas-protein (Cas9) nuclease, guide RNA (gRNA), and a defined 20 nucleotide site, CRISPR can induce specific cuts, or cleaves, within a DNA sequence. Infographic created by Daniel Alique García on https://plantae.org/crispr-vs-allergens/ Soybean—a major food crop in the United States—is known to be one of eight major allergens. Two proteins found within soybean are key contributors to allergens in humans: glycoprotein Gly m Bd 29 K and protein Gly m Bd 30 K (Sugano et al., 2020). To knock out genes encoding these two proteins, researchers utilized CRISPR-Cas9 systems along with Agrobacterium-mediated transformation. This gene knockout subsequently reduced protein expression in transgenic soybean plants when compared to original, wildtype soybean, proving initial success for future food allergy-related endeavors (Sugano et al., 2020). A study with peanuts and the Ara h 2 glycoprotein utilized a technology similar to CRISPR (RNA interference, or RNAi) to induce a knock-down gene expression of this glycoprotein which plays a role in peanut allergenicity (Dodo et al., 2007). The RNAi plasmid was then delivered to peanut plants and integrated into the plant’s genome. A 25% decrease in Ara h 2 glycoprotein was observed in seeds from transgenic plants compared to the original peanut. This means that the new, transgenic plant is not completely rid of the peanut allergen, but the reduction of Ara h 2 is certainly a research advancement. There is still major research that must be conducted to fully eliminate IgE food allergens from food crops. However, this research is a fundamental step in the right direction and will hopefully give people an allergy-free future! References Dodo, H. W., Konan, K. N., Chen, F. C., Egnin, M., & Viquez, O. M. (2008). Alleviating peanut allergy using genetic engineering: the silencing of the immunodominant allergen Ara h 2 leads to its significant reduction and a decrease in peanut allergenicity. Plant biotechnology journal, 6(2), 135-145. FDA (2023, December 08). FDA Approves First Gene Therapies to Treat Patients with Sickle Cell Disease https://www.fda.gov/news-events/press-announcements/fda-approves-first-gene-therapies -treat-patients-sickle-cell-disease Vos, T., Barber, R. M., Bell, B., Bertozzi-Villa, A., Biryukov, S., Bolliger, I., ... & Brugha, T. S. (2015). Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990–2013: a systematic analysis for the Global Burden of Disease Study 2013. The lancet, 386(9995), 743-800. Gupta, R. S., Warren, C. M., Smith, B. M., Jiang, J., Blumenstock, J. A., Davis, M. M., ... & Nadeau, K. C. (2019). Prevalence and severity of food allergies among US adults. JAMA network open, 2(1), e185630-e185630. Sugano, S., Hirose, A., Kanazashi, Y., Adachi, K., Hibara, M., Itoh, T., ... & Yamada, T. (2020). Simultaneous induction of mutant alleles of two allergenic genes in soybean by using site-directed mutagenesis. BMC Plant Biology, 20, 1-15. Kyla Danae Hughes is a rising Sophomore within the College of Agriculture at Tennessee State University. Kyla is a farm bill scholar and a Dean's scholar involved in research projects on CRISPR-cas use in the legume Medicago truncatula. Kyla loves spending time outdoors, hiking and backpacking. She is also an accomplished violinist with a passion for baking desserts!