VEGETABLE CROP IMPROVEMENT VIA CRISPR/CAS-9 MEDIATED GENOME EDITING

Abstract: The rapidly growing global population leads to an increase in food demand. Vegetables are essential for meeting the daily dietary needs of humans, as they are good sources of fiber, vitamins, and macro as well as micronutrients. Deficiencies of these nutrients are a major health concern, necessitating increased production and the development of new varieties having higher yield and high nutritive value. Conventional breeding methods used in vegetable crop improvement are time consuming and highly laborious, causing delays in the development of new varieties. To overcome the constraints of traditional breeding methods, CRISPR/Cas-9 (Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR-Associated Protein-9) is an advanced genome-editing technology offering more accurate results in less time as compared with the conventional methods. This technology helps crop breeders to insert, delete, or change specific sequences of DNA for the modification of the genome. The system uses the Cas9 endonuclease in combination with a single guide RNA (sgRNA) to recognize complementary genomic sequences and induce targeted double strand breaks (DSBs). These double-strand breaks are repaired by different cellular mechanisms, mostly by Non-Homologous End Joining (NHEJ) or by Homology Directed Repair (HDR), they enables nucleotide insertions, deletions, or substitutions that help in targeted genetic modification. CRISPR/Cas-9 is currently used in vegetable crop improvement programmes to introduce resistance against both biotic (insects, pests, diseases, etc) as well as abiotic stresses (heat, drought, salinity, etc). For example, mutations induced by CRISPR/Cas-9 in SlMAPK3 enhance heat and drought tolerance in tomato. Resistance against powdery mildew in tomato is achieved by knocking out the S1Mlo1 gene. Despite its potential, challenges remain, including the crucial need to select the correct target gene and mutation type so as to avoid off-target editing, and difficulties associated with the transfer of the gene-editing agents into plant cells. However, its precision and speed, particularly in improving polygenic traits, CRISPR/Cas-9 holds vast future scope and is said to be a revolution in vegetable crop breeding and biotechnology.

Keywords: CRISPR/ Cas-9, Vegetable Breeding, Technology, Speed Breeding, Generate editing.

Author: Shivanshu Bhalwal, Haseeb Khan, Riya Guptaand Kajal Sharma

Reference: Chandrasekaran, J., Brumin, M., Wolf, D., Leibman, D., Klap, C., Pearlsman, M., et al. (2016). Development of broad virus resistance in non-transgenic Cucumber using CRISPR/Cas9 technology. Mol. Plant Pathol. 17, 1140–1153. Doi: 10.1111/mpp.12375 Devi, R., Chauhan, S., & Dhillon, T. S. (2022). Genome editing for vegetable crop improvement: Challenges and future prospects. Frontiers in Genetics, 13, 1037091. Doudna, J. A., and Charpentier, E. (2014). Genome editing. The new frontier of Genome engineering with CRISPR-Cas9. Science 346, Doi:10.1126/science. 1258096 1258096. Das, T., Anand, U., Pal, T., Mandal, S., Kumar, M., Radha, … & Dey, A. (2023). Exploring the potential of CRISPR/Cas genome editing for vegetable crop improvement: an overview of challenges and approaches. Biotechnology and 120(5), 1215-1228. Bioengineering, Gasiunas, G., Barrangou, R., Horvath, P., and Siksnys, V. (2012). Cas9-crRNA Ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity In bacteria. Proc. Natl. Acad. Sci. U. S. A. 109, E2579–E2586. Doi:10.1073/pnas. 1208507109 Huang, Y., Cao, H., Yang, L., Chen, C., Shabala, L., Xiong, M., et al. (2019). Tissue- Specific respiratory burst oxidase homolog-dependent H2O2 signaling to the plasma Membrane H+-ATPase confers potassium uptake and salinity tolerance inCucurbitaceae. J. Exp. Bot. 70, 5879–5893. Doi:10.1093/jxb/erz328 Haque, E., Taniguchi, H., Hassan, M., Bhowmik, P., Karim, M. R., Smiech, M., Et al. (2018). Application of CRISPR/Cas9 genome editing technology for the Improvement of crops cultivated in tropical climates: Recent progress, prospects, And challenges. Front. Plt. Sci. 9, Doi:10.3389/fpls.2018.00617d 617. Jeong, S. Y., Ahn, H., Ryu, J., Oh, Y., Sivanandhan, G., Won, K. H., et al. (2019). Generation of early-flowering Chinese cabbage (Brassica rapa spp. Pekinensis). Through CRISPR/Cas9 mediated genome editing. Plant Biotechnol. Rep. 13, 491–499. Doi:10.1007/s11816-019-00566-9 Klimek-Chodacka, M., Oleszkiewicz, T., Lowder, L. G., Qi, Y., and Baransk, R. (2018). Efficient CRISPR/Cas9-based genome editing in carrot cells. Plant Cell Rep. 37, 575–586. Doi:10.1007/s00299 018-2252-2 Karkute, S. G., Singh, A. K., Gupta, O. P., Singh, P. M., & Singh, B. (2017). CRISPR/Cas9 engineering mediated for genome improvement of horticultural crops. Frontiers in plant science, 8, 1635. Liu, M., Rehman, S., Tang, X., Gu, K., Fan, Q., Chen, D., et al. (2019). Methodologies for improving HDR efficiency. Front. Genet. 9, 691–699. Doi:10. 3389/fgene.2018.0069 Makhotenko, A. V., Khromov, A. V., Snigir, E. A., Makarova, S. S., Makarov, V. V., Suprunova, T. P., et al. (2019). Functional analysis of coilin in virus resistance And stress tolerance of potato Solanum tuberosum using CRISPR-cas9 editing. Dokl. Biochem. Biophys. 484, 88-91.Doi:10.1134/S1607672919010241 Nakayasu, M., Akiyama, R., Lee, J. H., Osakabe, K., Osakabe, Y., Watanabe, B.,Et al. (2018). Generation of α solanine-free hairy roots of potato by CRISPR/ Cas9 mediated genome editing of the St16DOX gene. Plant Physiol. 6

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