Advancements in CRISPR-Mediated Multiplex Genome Editing: Transforming Plant Breeding for Crop Improvement and Polygenic Trait Engineering

The advent of CRISPR/Cas systems has revolutionized plant genome engineering, transitioning from traditional single-gene edits to sophisticated multiplex genome editing strategies capable of simultaneously targeting multiple loci. This review provides an in-depth examination of CRISPR-mediated multiplexing technologies in plants, emphasizing their molecular mechanisms, delivery systems, and transformative applications in crop improvement. We delineate the evolution of CRISPR systems from early programmable nucleases to diverse Class 2 effectors, including Cas9, Cas12, Cas13, and emerging ultra-compact variants like CasΦ and Cas14. We detail polycistronic gRNA expression platforms—such as tRNA-sgRNA arrays, ribozymes, and Csy4-mediated cleavage—that enable efficient multi-target editing within compact vectors. Furthermore, we explore advanced delivery modalities including Agrobacterium, biolistics, protoplast transfection, and viral vectors, optimized for recalcitrant plant systems. Applications span yield enhancement, disease resistance, abiotic stress tolerance, nutritional fortification, and de novo domestication. Critical challenges including off-target mutagenesis, mosaicism, chromosomal rearrangements, and regulatory constraints are addressed. Finally, we highlight AI-driven sgRNA design, multi-omics integration, and CRISPR libraries as pivotal tools to rationalize and scale multiplex editing. This synthesis underscores multiplex CRISPR as a cornerstone of next-generation plant breeding, with the potential to redefine global agriculture through precision trait stacking and rapid varietal development.