Genome editing beyond transgenes: DNA-free technologies in crop improvement.

Conventional and transgenic approaches to crop improvement are increasingly constrained by biosafety concerns, stringent regulatory frameworks, and limited public acceptance, necessitating new precision breeding methods. DNA-free genome editing using the CRISPR/Cas9 approach has emerged as a transformative approach that enables precise, targeted genetic modifications without the stable integration of a foreign gene (transgene). This strategy transiently introduces CRISPR/Cas9 ribonucleoprotein (RNP) complexes, or in vitro transcribed mRNA, into plant cells. This review focuses primarily on CRISPR/Cas-mediated DNA-free genome editing and its applications in crop improvement. After targeted genome cleavage, the editing elements are rapidly degraded, leaving only the desired genomic modification and resulting in plants indistinguishable from those developed through conventional breeding. To achieve targeted, DNA-free genome editing, researchers have developed numerous molecular platforms and delivery systems, such as protoplast transfection, biolistic bombardment, nanoparticle-based systems, and emerging pollen- and meristem-targeted systems, for major crop species. Researchers have successfully enhanced several notable traits, including disease and pest resistance, nutritional quality, abiotic stress tolerance, and agronomic performance, in cereals, vegetables, oilseeds, and perennial crops. Recent advancements, including base editing, prime editing, and multiplex gene editing, are further expanding the precision, efficiency, and scope of DNA-free genome editing. Overall, CRISPR/Cas-mediated DNA-free genome editing offers a route to crop improvement that avoids stable transgene integration, though its wider deployment depends on progress in regeneration, molecular validation, and regulatory convergence.