Plant and Food Carotenoids pp 321-341 | Cite as
CRISPR-Cas-Mediated Gene Knockout in Tomato
Protocol
First Online:
Abstract
Loss-of-function mutants are crucial for plant functional genomics studies. With the advent of CRISPR-Cas genome editing, generating null alleles for one or multiple specific gene(s) has become feasible for many plant species including tomato (Solanum lycopersicum). An easily programmable RNA-guided Cas endonuclease efficiently creates DNA double-strand breaks (DSBs) at targeted genomic sites that can be repaired by nonhomologous end joining (NHEJ) typically leading to small insertions or deletions that can produce null mutations. Here, we describe how to utilize CRISPR-Cas genome editing to obtain stable tomato gene knockout lines.
Key words
Genome editing CRISRP-Cas Gene knockout Loss-of-function mutation Null mutation Site-directed mutagenesis Targeted mutagenesis Tomato Solanum lycopersicum SolanaceaeReferences
- 1.Rothan C, Bres C, Garcia V, Just D (2016) Tomato resources for functional genomics. In: Causse M, Giovannoni J, Bouzayen M, Zouine M (eds) The tomato genome. Springer, Berlin, Germany, pp 75–94CrossRefGoogle Scholar
- 2.Van Eck J (2018) Genome editing and plant transformation of solanaceous food crops. Curr Opin Biotechnol 49:35–41CrossRefGoogle Scholar
- 3.Ron M, Kajala K, Pauluzzi G, Wang D, Reynoso MA, Zumstein K et al (2014) Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model. Plant Physiol 166:455–469CrossRefGoogle Scholar
- 4.Van Eck J, Keen P, Tjahjadi M (2019) Agrobacterium tumefaciens-mediated transformation of tomato. In: Kumar S, Barone P and Smith M (Eds) Transgenic plants (methods in molecular biology 1864). Humana Press, New York, NY, pp 225–234Google Scholar
- 5.Shikata M, Ezura H (2016) Micro-tom tomato as an alternative plant model system: mutant collection and efficient transformation. In: Botella J, Botella M (eds) Plant signal transduction (methods in molecular biology 1363). Humana Press, New York, NY, pp 47–55CrossRefGoogle Scholar
- 6.Garcia D, Narváez-Vásquez J, Orozco-Cárdenas ML (2015) Tomato (Solanum lycopersicum). In: Wang K (ed) Agrobacterium protocols (methods in molecular biology 1223). Springer, New York, NY, pp 349–361CrossRefGoogle Scholar
- 7.Lampropoulos A, Sutikovic Z, Wenzl C, Maegele I, Lohmann JU, Forner J (2013) GreenGate—a novel, versatile, and efficient cloning system for plant transgenesis. PLoS One 8:e83043CrossRefGoogle Scholar
- 8.Schiml S, Fauser F, Puchta H (2017) CRISPR/Cas-mediated in planta gene targeting. In: Busch W (ed) Plant genomics (methods in molecular biology 1610). Humana Press, New York, NY, pp 3–11Google Scholar
- 9.Ellul P, Garcia-Sogo B, Pineda B, Ríos G, Roig L, Moreno V (2003) The ploidy level of transgenic plants in Agrobacterium-mediated transformation of tomato cotyledons (Lycopersicon esculentum L. mill.) is genotype and procedure dependent. Theor Appl Genet 106:231–238CrossRefGoogle Scholar
- 10.Decaestecker W, Buono RA, Pfeiffer ML, Vangheluwe N, Jourquin J, Karimi M et al (2018) CRISPR-TSKO facilitates efficient cell type-, tissue-, or organ-specific mutagenesis in Arabidopsis. bioRxiv. 474981. https://doi.org/10.1101/474981
- 11.Popp MW, Maquat LE (2016) Leveraging rules of nonsense-mediated mRNA decay for genome engineering and personalized medicine. Cell 165:1319–1322CrossRefGoogle Scholar
- 12.Zischewski J, Fischer R, Bortesi L (2017) Detection of on-target and off-target mutations generated by CRISPR/Cas9 and other sequence-specific nucleases. Biotechnol Adv 35:95–104CrossRefGoogle Scholar
- 13.Jacobs TB, Martin GB (2016) High-throughput CRISPR vector construction and characterization of DNA modifications by generation of tomato hairy roots. J Vis Exp 110:e53843Google Scholar
- 14.Fauser F, Schiml S, Puchta H (2014) Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J 79:348–359CrossRefGoogle Scholar
- 15.Ritter A, Iñigo S, Fernández-Calvo P, Heyndrickx KS, Dhondt S, Shi H et al (2017) The transcriptional repressor complex FRS7-FRS12 regulates flowering time and growth in Arabidopsis. Nat Commun 8:15235CrossRefGoogle Scholar
- 16.Pauwels L, De Clercq R, Goossens J, Iñigo S, Williams C, Ron M et al (2018) A dual sgRNA approach for functional genomics in Arabidopsis thaliana. G3 Genes 8:2603–2615Google Scholar
- 17.Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z et al (2015) Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523:481–485CrossRefGoogle Scholar
- 18.Houbaert A, Zhang C, Tiwari M, Wang K, de Marcos Serrano A, Savatin DV et al (2018) POLAR-guided signalling complex assembly and localization drive asymmetric cell division. Nature 563:574–578CrossRefGoogle Scholar
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