Application of CRISPR/Cas9-Mediated Gene Editing in Tomato
CRISPR-/Cas9-mediated gene editing has been demonstrated in a number of food crops including tomato. Tomato (Solanum lycopersicum) is both an important food crop and a model plant species that has been used extensively for studying gene function, especially as it relates to fruit biology. This duality in purpose combined with readily available resources (mutant populations, genome sequences, transformation methodology) makes tomato an ideal candidate for gene editing. The CRISPR/Cas9 system routinely used in our laboratory has been applied to various tomato genotypes and the wild species, Solanum pimpinellifolium. The vector system is based on Golden Gate cloning techniques. Cassettes that contain the neomycin phosphotransferase II (NPTII) selectable marker gene that confers resistance to kanamycin, a human codon-optimized Cas9 driven by the CaMV 35S promoter, and guide RNA (gRNA) under control of the Arabidopsis U6 polymerase promoter are assembled into a T-DNA vector. Generally, we design CRISPR/Cas9 constructs that contain two gRNAs per gene target. However, we have been successful with inclusion of up to eight gRNAs to simultaneously target multiple genes and regions. Introduction of CRISPR-/Cas9-designed constructs into tomato is accomplished by transformation methodology based on Agrobacterium tumefaciens infection of young cotyledon sections and selection on kanamycin-containing medium based on the presence of the NPTII gene. The approaches for the development of CRISPR/Cas9 constructs and genotypic analyses (PCR-based amplicon sequencing and T7 endonuclease) are detailed in this chapter.
Key wordsGenome editing CRISPR/Cas9 PCR amplicon sequencing Solanaceae Solanum lycopersicum Solanum pimpinellifolium T7 endonuclease 1 (T7E1) assay
The authors acknowledge the National Science Foundation Plant Genome Research Program (IOS-1732253) for support related to their research on gene editing in tomato. The authors would like to thank Sophien Kamoun and Vlad Nekrasov for the guidance during the early development of gene editing methodology for tomato with their vector system. The authors also acknowledge the members of the Lippman lab at Cold Spring Harbor Laboratory and members of the Van Eck lab affiliated with tomato transformation. Michelle Tjahjadi provided the flowchart included in Fig. 1.
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