Lycopene Is Enriched in Tomato Fruit by CRISPR/Cas9-Mediated Multiplex Genome Editing - PubMed (original) (raw)
Lycopene Is Enriched in Tomato Fruit by CRISPR/Cas9-Mediated Multiplex Genome Editing
Xindi Li et al. Front Plant Sci. 2018.
Abstract
Numerous studies have been focusing on breeding tomato plants with enhanced lycopene accumulation, considering its positive effects of fruits on the visual and functional properties. In this study, we used a bidirectional strategy: promoting the biosynthesis of lycopene, while inhibiting the conversion from lycopene to β- and α-carotene. The accumulation of lycopene was promoted by knocking down some genes associated with the carotenoid metabolic pathway. Finally, five genes were selected to be edited in genome by CRISPR/Cas9 system using _Agrobacterium tumefaciens_-mediated transformation. Our findings indicated that CRISPR/Cas9 is a site-specific genome editing technology that allows highly efficient target mutagenesis in multiple genes of interest. Surprisingly, the lycopene content in tomato fruit subjected to genome editing was successfully increased to about 5.1-fold. The homozygous mutations were stably transmitted to subsequent generations. Taken together, our results suggest that CRISPR/Cas9 system can be used for significantly improving lycopene content in tomato fruit with advantages such as high efficiency, rare off-target mutations, and stable heredity.
Keywords: CRISPR/Cas9 system; carotenoid metabolic pathway; genome editing; lycopene; tomato fruits.
Figures
FIGURE 1
Selection of target genes and designing of CRISPR/Cas9 binary expression cassette. (A) A map of the target genes in the carotenoid metabolic pathway. The green boxes represent the key substances in the metabolic pathway. The red and orange boxes show the two substances, lycopene and β-carotene, respectively. A solid arrow indicates a direct effect, and a dashed arrow indicates an indirect effect. The selected target genes are represented by purple boxes, and the red asterisks represent the sites at which the target genes act on the pathway. G3P, glyceraldehyde 3-phosphate; DXS, 1-deoxy-
D
-xylulose 5-phosphate synthase; GGPPS, geranylgeranyl pyrophosphate synthase; PDS, phytoene desaturase; ZISO, z-carotene isomerase. (B) Five target genes were selected according to the synthesis and metabolism pathways of lycopene, and six target sites were designed. The target sequences are marked in red, and small rectangle frames indicate the PAM. Straight lines and boxes are the introns and exons of the target genes, respectively. (C) Structures of the pYLCRISPR/Cas9-Lycopene binary vectors. HPT(-H) encodes hygromycin B phosphotransferase. The six targets designed are represented by solid boxes in different colors, and the promoters used for each target are shown.
FIGURE 2
Editing of each target site in the pYLCRISPR/Cas9-lycopene expression cassette. (A) Total editing efficiency of the six targets in the pYLCRISPR/Cas9-lycopene expression cassette. The mutation rate is the ratio of the number of mutations detected to that of the total number of plants in which mutations are detected. (B) Specific types of each target among the six target sites in the pYLCRISPR/Cas9-lycopene expression cassette. Green, orange, purple, and blue represent homozygous, biallelic, heterozygous, and chimeric mutations, respectively. (C) Large fragment deletion on Blc. Target sequence is labeled with yellow. Small rectangle frames indicate the protospacer adjacent motifs. The transcriptional direction of the Blc is shown. (D) Special DNA inversion on SGR1. The red and blue parts represent the targets T1 and T2, respectively. Gradient change of color between yellow and white indicates the transcriptional direction of SGR1.
FIGURE 3
Determination of lycopene content in tomato fruit at Br+7 from different mutant groups. (A) List of different groups of lycopene mutants according to the different combinations of mutant genes. (B) HPLC results of crude extracts from tomato fruit samples in different groups. a, b, lycopene and β-carotenoids, respectively. (C,D) Contents of lycopene and β-carotenoids of tomato fruit in the five different groups and WT, respectively. Error bars represent standard deviation. Different lowercase letters show statistically significant difference according to ANOVA followed by Duncan’s test (p < 0.05).
FIGURE 4
TEM images of epidermal cells in tomato fruit of WT and lycopene mutants at Br+7. c, carotenoid containing structures; e, plastid envelope; l, crystal line. Bar, 1 μm.
FIGURE 5
Phenotype of tomato fruit at different ripening stages. Three groups of transgenic tomato fruits were photographed at different times after the breaker stage of ripening and compared with that in WT. Sections were obtained at Br+7. Each group photograph was obtained from the same tomato fruit. e, exocarp; i, endocarp and f, flesh of the tomato fruit.
Similar articles
- CRISPR/Cas9 directed editing of lycopene epsilon-cyclase modulates metabolic flux for β-carotene biosynthesis in banana fruit.
Kaur N, Alok A, Shivani, Kumar P, Kaur N, Awasthi P, Chaturvedi S, Pandey P, Pandey A, Pandey AK, Tiwari S. Kaur N, et al. Metab Eng. 2020 May;59:76-86. doi: 10.1016/j.ymben.2020.01.008. Epub 2020 Jan 30. Metab Eng. 2020. PMID: 32006663 - CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations.
Pan C, Ye L, Qin L, Liu X, He Y, Wang J, Chen L, Lu G. Pan C, et al. Sci Rep. 2016 Apr 21;6:24765. doi: 10.1038/srep24765. Sci Rep. 2016. PMID: 27097775 Free PMC article. - CRISPR/Cas genome editing in tomato improvement: Advances and applications.
Tiwari JK, Singh AK, Behera TK. Tiwari JK, et al. Front Plant Sci. 2023 Feb 23;14:1121209. doi: 10.3389/fpls.2023.1121209. eCollection 2023. Front Plant Sci. 2023. PMID: 36909403 Free PMC article. Review. - Targeted editing of tomato carotenoid isomerase reveals the role of 5' UTR region in gene expression regulation.
Lakshmi Jayaraj K, Thulasidharan N, Antony A, John M, Augustine R, Chakravartty N, Sukumaran S, Uma Maheswari M, Abraham S, Thomas G, Lachagari VBR, Seshagiri S, Narayanan S, Kuriakose B. Lakshmi Jayaraj K, et al. Plant Cell Rep. 2021 Apr;40(4):621-635. doi: 10.1007/s00299-020-02659-0. Epub 2021 Jan 15. Plant Cell Rep. 2021. PMID: 33449143 - Multiplex Genome Editing in Yeast by CRISPR/Cas9 - A Potent and Agile Tool to Reconstruct Complex Metabolic Pathways.
Utomo JC, Hodgins CL, Ro DK. Utomo JC, et al. Front Plant Sci. 2021 Aug 5;12:719148. doi: 10.3389/fpls.2021.719148. eCollection 2021. Front Plant Sci. 2021. PMID: 34421973 Free PMC article. Review.
Cited by
- Intra-canopy LED lighting outperformed top LED lighting in improving tomato yield and expression of the genes responsible for lycopene, phytoene and vitamin C synthesis.
Ziaei N, Talebi M, Sayed Tabatabaei BE, Sabzalian MR, Soleimani M. Ziaei N, et al. Sci Rep. 2024 Aug 16;14(1):19043. doi: 10.1038/s41598-024-69210-z. Sci Rep. 2024. PMID: 39152138 Free PMC article. - A Comprehensive Protocol for Assembly of Multiple gRNAs into a Direct Vector for Genome Editing in Tomato.
Satyavathi VV, Princy K, Gupta N, Nizampatnam NR, Sharma R, Sreelakshmi Y. Satyavathi VV, et al. Methods Mol Biol. 2024;2788:317-335. doi: 10.1007/978-1-0716-3782-1_19. Methods Mol Biol. 2024. PMID: 38656523 - Enhancement of specialized metabolites using CRISPR/Cas gene editing technology in medicinal plants.
Das S, Kwon M, Kim JY. Das S, et al. Front Plant Sci. 2024 Feb 21;15:1279738. doi: 10.3389/fpls.2024.1279738. eCollection 2024. Front Plant Sci. 2024. PMID: 38450402 Free PMC article. Review. - New Advances in the Study of Regulation of Tomato Flowering-Related Genes Using Biotechnological Approaches.
Baranov D, Dolgov S, Timerbaev V. Baranov D, et al. Plants (Basel). 2024 Jan 25;13(3):359. doi: 10.3390/plants13030359. Plants (Basel). 2024. PMID: 38337892 Free PMC article. Review. - Generation of parthenocarpic tomato plants in multiple elite cultivars using the CRISPR/Cas9 system.
Nguyen CC, Van Vu T, Shelake RM, Nguyen NT, Khanh TD, Kim WY, Kim JY. Nguyen CC, et al. Mol Breed. 2024 Feb 3;44(2):13. doi: 10.1007/s11032-024-01452-1. eCollection 2024 Feb. Mol Breed. 2024. PMID: 38317771
References
- Barry C. S., McQuinn R. P., Chung M. Y., Besuden A., Giovannoni J. J. (2008). Amino acid substitutions in homologs of the STAY-GREEN protein are responsible for the green-flesh and chlorophyll retainer mutations of tomato and pepper. Plant Physiol. 147 179–187. 10.1104/pp.108.118430 - DOI - PMC - PubMed
LinkOut - more resources
Full Text Sources
Other Literature Sources