Abstract
CRISPR-Act3.0 is a robust tool for modulating fatty acid profiles in plants. We demonstrate that Agrobacterium rhizogenes-mediated transformation provides a rapid, cost-effective, and equipment-independent platform for validating sgRNA efficiency and metabolic outcomes within a short time. The CRISPR-Act3.0 system offers a powerful strategy for activating endogenous gene expression in plants. However, the labor-intensive and time-consuming nature of stable transformation often hinders the rapid validation of multiple sgRNAs. In this study, we optimized a rapid Agrobacterium rhizogenes-mediated transient expression system in tomato to evaluate sgRNA efficiency within the CRISPR-Act3.0 framework. As a proof-of-concept, we targeted four genes involved in fatty acid biosynthesis: SlFATA, SlFATB-01, SlFATB-02, and SlFATB-03. To ensure precise control, we utilized the root-specific pSMB promoter to drive the CRISPRa components. Our results demonstrate that this system can successfully induce significant transcriptional activation and alter fatty acid compositions specifically increasing palmitic acid levels by up to 45%-within approximately 30 days. This approach bypasses the requirement for whole-plant stable transformation during the initial screening phase and prevents potential pleiotropic effects by restricting activation to root tissues. Overall, this study provides a highly efficient diagnostic pipeline for functional genomics and metabolic engineering in tomato, offering a significant advantage for rapid trait evaluation before committing to stable transgenic line production.