![]() ![]() The application of CRISPR-Cas9 in tomato genome engineering obtained targeted mutations at four different loci. Therefore, new tomato cultivars capable of biotic and abiotic stress tolerance are important subjects of breeding using GE technology. Recently, unforeseen climate changes have caused many biotic and abiotic stresses that negatively affect multiple aspects of tomato production, such as yield and quality, thereby threatening agriculture sustainability. It has been successful in modifying tomato genomes and precision breeding to create new alleles, not including linkage drag ( Zsögön et al., 2017 Li et al., 2018b Zsögön et al., 2018 Vu et al., 2020a). CRISPR-Cas-based GE has been effectively applied for plant breeding at high precision levels ( Chen et al., 2019). Genome editing (GE) technology using CRISPR-Cas nucleases has recently emerged as a revolutionary plant breeding technology and shows promise to the agriculture industry ( Belhaj et al., 2013 Chen et al., 2019 Vu et al., 2022). CRISPR-Cas9-based precise domain editing of the SlHyPRP1 gene generated multi-stress-tolerant alleles that could be used as genetic materials for tomato breeding. However, the edited alleles enhanced susceptibility against Fusarium oxysporum f. tomato ( Pto) DC3000, the growth of the bacterium was significantly reduced by 2.0- to 2.5-fold compared to that in WT plants. When the edited lines were challenged with pathogenic bacteria of Pseudomonas syringae pv. Moreover, the PR1v1 line continuously grew after 5 days of water cutoff. Similarly, plants carrying either the domains of the PRD removal variant (PR1v1) or 8CM removal variants (PR2v2 and PR2v3) showed better germination under osmosis stress (up to 200 mM mannitol) compared to the WT control. Our data reveal that the 8CM removal variants of HK and the KO alleles of both HK and 15T01 cultivars exhibited moderate heat stress tolerance. In this study, we characterized the edited lines under several abiotic and biotic stresses to examine the possibility of multiple stress tolerance. We subsequently demonstrated that eliminating the PRD domain of HyPRP1 in tomatoes conferred the highest level of salinity tolerance. In our previous report, a tomato gene encoding hybrid proline-rich protein 1 (HyPRP1), a negative regulator of salt stress responses, has been edited using a CRISPR-Cas9 multiplexing approach that resulted in precise eliminations of its functional domains, proline-rich domain (PRD) and eight cysteine-motif (8CM). Recently, CRISPR-Cas9-based genome editing has been widely used for plant breeding. 6Nulla Bio R&D Center, Nulla Bio Inc., Jinju, Republic of Korea.5National Key Laboratory for Plant Cell Biotechnology, Agricultural Genetics Institute, Hanoi, Vietnam.4Division of Life Science, Gyeongsang National University, Jinju, Republic of Korea.3Division of Horticultural Science, Gyeongsang National University, Jinju, Republic of Korea. ![]() 2Crop Science and Rural Development Division, College of Agriculture, Bac Lieu University, Bac Lieu, Vietnam.1Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, Republic of Korea.The pasted sequence should be FASTA format.Mil Thi Tran 1,2,3†, Geon Hui Son 1†, Young Jong Song 1†, Ngan Thi Nguyen 1, Seonyeong Park 1, Thanh Vu Thach 1, Jihae Kim 1, Yeon Woo Sung 1, Swati Das 1, Dibyajyoti Pramanik 1, Jinsu Lee 1, Ki-Ho Son 3, Sang Hee Kim 1,4*‡, Tien Van Vu 1,5*‡ and Jae-Yean Kim 1,4,6*‡ Please choose the targets to test and/or paste a custom sequence into the text area below. Select to check for secondary off-targets Tolerated edit distance to the target sequenceĬheck off-targets in non-gene regions should be ignored Retrieve and save a recombination donor matrixĪssess restriction sites of the whole sequence Įxclude designs with more than X off-targets Number of coding exons downstream the start codon for K.O.īp Minimal spacer length for paired designsīp Maximum spacer length for paired designs Select if CRISPR designs should be used for tagging or knockout experimentsīp Minimum guide RNA length after PAM īp Maximum guide RNA length after PAM īp Tagging window downstream of the codon (only NGG PAM, only G as 5' base, off-target tolerates many mismatches and ignores non-seed region, introns, purpose is knockout (only first 3 coding exons are allowed) and UTRs are excluded) (any PAM (NAG/NGG.), any 5' base (A,C,G,T.), off-targets tolerate mismatches, introns/CPG islands are excluded) (any PAM (NAG/NGG.), any 5' base (A,C,G,T.), off-targets need full length perfect match, introns are allowed) ![]()
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