| 초록 |
□ 연구개발 목표 및 내용 ◼ 최종 목표 The project’s ultimate goal is to generate multi-stress tolerant tomato plants using CRISPR-Cas9-based precise gene engineering of SlHyPRP1, a multi-stress response protein. The following works were done to achieve the goal: 1, Designed multiplexed CRISPR-Cas9 constructs for generating dual DNA double-stranded breaks (DSBs) for deleting either the domains of SlHyPRP1. 2. Transformed the gene editing constructs into tomato using Agrobacterium-mediated transformation method and screened for precisely edited alleles by molecular and sequencing methods. 3. Raised plants carrying homozygously edited alleles and free of transgene up to the third generation. 4. Evaluated the possibility of multi-stress tolerance. ◼ 전체 내용 Environmental stresses threaten global food crop production while arable land is reducing and the population is increasing. Under the circumstance, stress-tolerant, especially multi-stress tolerant crops are highly demanded to sustain food production. Multi-stress tolerance could be achieved by engineering genes involved in multi-stress responses as a negative regulator. The proline-rich (PRD) and eight cysteine motif (8-CM) domains-carrying SlHyPRP1 protein containing was shown to participate in multi-stress responses as either a negative or positive regulator. Precise genome modification at the gene/locus scale was nearly impossible for plant breeding using conventional methods. Recently, the cutting-edge CRISPR-Cas9 technology has enabled precise gene editing down to every single base scale and is thus being applied for generating precise gene engineering in plants. In this project, CRISPR-Cas9 was used as a molecular scissor for generating a pair of blunt-end DSBs at the boundary of the SlHyPRP1’s domains, thereby forming three types of alleles: 1, PRD-removal; 2, 8-CM removal; and 3, knockout (KO). The precise deletion of the domain was mediated by the canonical non-homologous end-joining (cNHEJ) that rejoins the blunt ends of the DSBs with the possibility of omitting the DNA fragment residing between the DSB sites. Up to the third generation of the HyPRP1-edited lines were raised for obtaining plants carrying homozygously edited alleles and also free of transgenes. The homozygous plants were first subjected to salinity stress conditions, resulting in high salt tolerance of the plants missing the PRD domain (only the 8-CM or knockout allele). Subsequently, the edited lines were challenged with various stresses, including abiotic (heat, osmosis, and drought) and biotic (plant-pathogenic bacteria and fungi). The obtained data revealed that some edited alleles supported multi-stress tolerance. The technology and approach can be applied for breeding multi-stress tolerant crops. □ 연구개발성과 In the frame of this project, we published 06 Q1 SCI (total IF2021=41.647) and 01 non-SCI paper. The data obtained from this work were also used to apply for one patent (10-2021-0139028). In addition, one patent application (10-2021-0089814) was supported by this project. Papers Tran, M. T., Son, G. H., Song, Y. J., Nguyen, N. T., Park, S., Thach, T. V., Kim, J., Sung, Y. W., Das, S., Pramanik, D., Lee, J., Son, K., Kim, S. H., Vu, T. V., & Kim, J. (2023). CRISPR-Cas9-based precise engineering of SlHyPRP1 protein towards multi-stress tolerance in tomato. Frontiers in Plant Science, 14. https://doi.org/10.3389/fpls.2023.1186932. Tien Van Vu, Ngan Thi Nguyen, Jihae Kim, Swati Das, Jinsu Lee, Jae-Yean Kim (2020) The Obstacles and Potential Solution Clues of Prime Editing Applications in Tomato. BioDesign Res. 2022;0001. DOI:10.34133/bdr.0001. Van Vu, T., Das, S., Hensel, G. et al. Genome editing and beyond: what does it mean for the future of plant breeding?. Planta 255, 130 (2022). https://doi.org/10.1007/s00425-022-03906-2 Vu, T. V., Das, S., Nguyen, C. C., Kim, J., & Kim, J.-Y. (2022). Single-strand annealing: molecular mechanisms and potential applications in CRISPR-Cas-based precision genome editing. Biotechnology Journal, 17, e2100413. https://doi.org/10.1002/biot.202100413. Vu, T. V |
