Browse > Article
http://dx.doi.org/10.5338/KJEA.2021.40.3.21

Evaluation of sgRNAs Targeting Pectate Lyase and Phytoene Synthase for Delaying Tomato Fruit Ripening  

Park, Hyosun (Department of Agricultural Chemistry, Chonnam National University)
Yang, So Hee (Department of Agricultural Chemistry, Chonnam National University)
Kim, Euyeon (Department of Agricultural Chemistry, Chonnam National University)
Koo, Yeonjong (Department of Agricultural Chemistry, Chonnam National University)
Publication Information
Korean Journal of Environmental Agriculture / v.40, no.3, 2021 , pp. 179-185 More about this Journal
Abstract
BACKGROUND: Tomato genome editing using CRISPR-Cas9 is being actively conducted in recent days, and lots of plant researches have been aiming to develop high valued crops by editing target genes without inserting foreign genes. Many researchers have been involved in the manipulation of the crop ripening process because fruit ripening is an important fruit phenotype for increasing fruit shelf life, taste, and texture of crops. This paper intends to evaluate target sgRNA to edit the two ripening-related genes encoding pectate lyase (PL) and phytoene synthase (Psy) with the CRISPR-Cas9 system. METHODS AND RESULTS: The CRISPR-Cas9 expression vector was cloned to target the PL (Solyc03g111690), Psy1 (Solyc03g031860), and Psy2 (Solyc02g081330) genes, which are the ripening genes of tomatoes. Tomatoes injected with Agrobacterium containing the CRISPR-Cas9 expression vector were further cultured for 5 days and used to check gene editing efficiency. As a result of the target gene sequence analysis by the next generation sequencing method, gene editing efficiency was calculated, and the efficient target location was selected for the PL and Psy genes. CONCLUSION: Therefore, this study was aimed to establish target sgRNA data that could have higher efficiency of the CRISPR-Cas9 system to obtain the delayed ripening phenotype of tomato. The developed method and sgRNA information is expected to be utilized in the development of various crops to manage its ripening processes.
Keywords
CRISPR-Cas9; Fruit Ripening; sgRNA Efficiency; Tomato;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Dahan-Meir T, Filler-Hayut S, Melamed-Bessudo C, Bocobza S, Czosnek H, Aharoni A, Levy AA (2018) Efficient in planta gene targeting in tomato using geminiviral replicons and the CRISPR/Cas9 system. Plant Journal, 95(1), 5-16. https://doi.org/10.1111/tpj.13932.   DOI
2 Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiamg W et al. (2013) Multiplex genome engineering using CRISPR/Cas systems. Science, 339(6121), 819-823. https://doi.org/10.1126/science.1231143.   DOI
3 Xie K, Yang Y (2013) RNA-guided genome editing in plants using a CRISPR-Cas system. Molecular Plant, 6(6), 1975-1983. https://doi.org/10.1093/mp/sst119.   DOI
4 Wang R, da Rocha Tavano EC, Lammers M, Martinelli AP, Angenent GC, de Maagd RA (2019) Re-evaluation of transcription factor function in tomato fruit development and ripening with CRISPR/Cas9-mutagenesis. Scientific Reports, 9(1), 1696. https://doi.org/10.1038/s41598-018-38170-6.   DOI
5 Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science, 339(6121), 823-826. https://doi.org/10.1126/science.1232033.   DOI
6 Seymour GB, Ostergaard L, Chapman NH, Knapp S, Martin C (2013) Fruit development and ripening. Annual Review of Plant Biology, 64, 219-241. https://doi.org/10.1111/nph.16724.   DOI
7 Keegstra K (2010) Plant cell walls. Plant Physiology, 154(2), 483-486. https://doi.org/10.1104/pp.110.161240.   DOI
8 Wang D, Samsulrizal NH, Yan C, Allcock NS, Craigon J, Blanco-Ulate B, Ortega-Salazar I, Marcus SE, Bagheri HM et al. (2019) Characterization of CRISPR mutants targeting genes modulating pectin degradation in ripening tomato. Plant Physiology, 179(2), 544-557. https://doi.org/10.1104/pp.18.01187.   DOI
9 Giorio G, Stigliani AL, D'Ambrosio C (2008) Phytoene synthase genes in tomato (Solanumlycopersicum L.) new data on the structures, the deduced amino acid sequences and the expression patterns. Federation of European Biochemical Societies Journal, 275(3), 527-535. https://doi.org/10.1111/j.1742-4658.2007.06219.x.   DOI
10 Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nature Protocols, 8(11), 2281-2308. https://doi.org/10.1038/nprot.2013.143.   DOI
11 Oleszkiewicz T, Klimek-Chodacka M, Kruczek M, Godel-Jedrychowska K, Sala K, Milewska-Hendel A, Zubko M, Kurczynska E, Qi Y et al. (2021) Inhibition of carotenoid biosynthesis by CRISPR/Cas9 triggers cell wall remodelling in carrot. International Journal of Molecular Sciences, 22(12), 6516. https://doi.org/10.3390/ijms22126516.   DOI
12 Ren F, Ren C, Zhang Z, Duan W, Lecourieux D, Li S, & Liang Z (2019) Efficiency optimization of CRISPR/Cas9-mediated targeted mutagenesis in grape. Frontiers in Plant Science, 10, 612. https://doi.org/10.3389/fpls.2019.00612.   DOI
13 Martin LB, Rose JK (2014) There's more than one way to skin a fruit: Formation and functions of fruit cuticles. Journal of Experimental Botany, 65(16), 4639-4651. https://doi.org/10.1093/jxb/eru301.   DOI
14 Uluisik S, Chapman NH, Smith R, Poole M, Adams G, Gillis RB, Besong TMD, Sheldon J, Stiegelmeyer S et al. (2016) Genetic improvement of tomato by targeted control of fruit softening. Nature Biotechnology, 34(9), 950-952. https://doi.org/10.1038/nbt.3602.   DOI
15 D'Ambrosio C, Stigliani AL, Giorio G (2018) CRISPR/Cas9 editing of carotenoid genes in tomato. Transgenic Research, 27(4), 367-378. https://doi.org/10.1007/s11248-018-0079-9.   DOI
16 Jung YJ, Bae SS, Lee GJ, Seo PJ, Cho YG, Kang KK (2017) A novel method for high-frequency genome editing in rice, using the CRISPR/Cas9 system. Journal of Plant Biotechnology, 44(1), 89-96. https://doi.org/10.5010/JPB.2017.44.1.089.   DOI
17 Lowder LG, Zhang D, Baltes NJ, Paul III J W, Tang X, Zheng X, Voytas DF, Hsieh TF, Zhang Y et al. (2015) A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiology, 169(2), 971-985. https://doi.org/10.1104/pp.15.00636.   DOI
18 Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Research, 19(6), 1349. https://doi.org/10.1093/nar/19.6.1349.   DOI