DNA-FREE EDITING VIA RNP DELIVERY IN OLIVE PROTOPLASTS

In recent years, remarkable progress in plant biotechnology has led to the emergence of powerful genome editing tools, revolutionizing the ability to manipulate plant genomes with precision and efficiency. These advancements have paved the way for innovative strategies aimed at enhancing crop traits, especially in species traditionally considered difficult to improve through conventional breeding or genetic transformation methods. Among these, the olive ( Olea europaea L.) stands out due to its economic and ecological relevance but also its notorious recalcitrance to in vitro regeneration and genetic transformation. The development of diverse genome editing strategies in plants has opened new avenues for the genetic improvement of such recalcitrant species, offering renewed hope for overcoming technical bottlenecks in olive biotechnology. This work aims to establish and optimize a DNA-free genome editing approach on olive protoplasts through the delivery of CRISPR/Cas9 ribonucleoprotein complexes (RNPs), targeting selected olive genes associated with Xylella fastidiosa susceptibility. Two main transfection methods are under evaluation: lipid-based delivery systems, specifically CRISPRMAX™ and LIPO3000™, and PEG-mediated transfection. Guide RNAs (gRNAs) are designed and synthesized in vitro to ensure high targeting specificity and editing efficiency, then pre-assembled with Cas9 prior to transfection. The success of this process is slightly dependent on the quality and yield of isolated protoplasts; to overcome this, an optimized isolation protocol is currently being developed using callus tissues from multiple olive genotypes grown in vitro, to maximize cell viability and editing competence. Despite the current limitation imposed by the low regeneration potential of protoplasts, mainly due to suboptimal culture conditions and reduced regeneration capacity, efforts will be addressed to evaluate the feasibility and efficiency of RNP-based genome editing in olive as a transgene-free strategy for genetic improvement.