ABSTRACT
The technical simplicity of CRISPR/Cas editing technology explains its rapid adoption in many laboratories, including small labs for which previous editing tools were inaccessible. Despite this, agrobacterium-mediated transformation remains the most popular approach for genome editing in plants, including many crops. Finally, we include a case study of the cloning of a nine gRNA multiplex construct and the analysis of its transformants in tobacco plants. In our laboratory we have developed a collection of CRISPR elements based on the GoldenBraid (GB) modular cloning system. This strategy, initially described in mammalian cells, was later adapted to plants and has subsequently been used in several demanding multiplexing approaches, successfully producing plants with several genes targeted simultaneously. In the context of improving tobacco plants as biofactories, the SPL-KO strategy aimed at generating new variability in growth habits, which could be later screened and selected for biofactory use.
