Targeted Gene Expression Modulation Using CRISPR/dCas9 to Investigate Pathogenic Outcomes in Tomato

CRISPR (clustered regularly interspaced short palindromic repeats) has become integral to modern biological research, with the Streptococcus pyogenes CRISPR/Cas9 system serving as the most extensively used tool for precise, site-specific genome editing across a wide range of organisms and cell types. Compared with earlier genome-editing platforms, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 offers greater simplicity, precision, versatility, and scalability. Beyond introducing stable DNA modifications, this system can be reengineered to reversibly activate (CRISPRa) or repress (CRISPRi) the transcription of any gene by employing unique nuclease-deactivated variants of Cas9 (dCas9) fused to transcriptional activators or repressors, respectively, providing a compelling alternative to RNA interference (RNAi) and conventional overexpression techniques. In plants, CRISPR/dCas9-based programmable gene control presents an innovative and transformative framework for rewiring gene regulatory networks to study pathogenic stress-signaling pathways. Notably, its strategic use in orchestrating the simultaneous regulation of multiple defense-related genes sets the stage for developing crops with robust and quantitative disease resistance. In this chapter, we outline a comprehensive methodology for the design, assembly, and functional assessment of CRISPR/dCas9 systems optimized for tomato to investigate pathogen-associated responses.