植物科学

Gene editing technologies have revolutionized plant molecular biology, providing powerful tools for precise gene manipulation for understanding function and enhancing or modifying agronomically relevant traits. Among these technologies, the CRISPR-Cas9 system has emerged as a versatile and widely accepted strategy for targeted gene manipulation. This protocol provides detailed, step-by-step instructions for implementing CRISPR-Cas9 genome editing in tomato plants, with a specific focus in generating knockout lines for a target gene. For that, the guide RNA should preferentially be designed within the first exon downstream and closer to the start codon. The edited plants obtained are free of transgene cassette for expression of the CRISPR-Cas9 machinery.

Somatic homologous recombination (SHR) is a major pathway of DNA double-strand break (DSB) repair, in which intact homologous regions are used as a template for the removal of lesions. Its frequency in plants is generally low, as most DSB are removed by non-homologous mechanisms in higher eukaryotes. Nevertheless, SHR frequency has been shown to increase in response to various chemical and physical agents that cause DNA damage and/or alter genome stability (reviewed in March-Díaz and Reyes, 2009). We monitor the frequency of SHR in transgenic Arabidopsis seedlings containing recombination substrates with two truncated but overlapping parts of the β-glucuronidase (GUS) reporter gene (Orel et al., 2003; Schuermann et al., 2005). Upon an SHR event, a functional version of the transgene can be restored (Figure 1A). A histochemical assay applicable to whole plantlets allows the visualization of cells in which the reporter is restored, as the encoded enzyme converts a colorless substrate into a blue compound. This type of reporter has been extensively used to identify gene products required for regulating SHR levels in plants. We analyze plants stimulated for SHR by treatments with DNA damaging agents (bleocin, mitomycin C and UV-C) and compare them to non-treated plants.