The generation of knock-out mutant lines to null the function of one or multiple genes is nowadays the requested quality standard to unequivocally resolve novel regulatory genetic pathways. CRISPR-Cas system has become the most straightforward tool in woody perennials research for this purpose. Moreover, the worldwide tendency regarding the regulation for the commercialization of products derived from genome-edited plants is evolving so that these plants are not considered GMOs when foreign DNA is removed. Considering this, together with the potential of targeted gene editing to improve any desired trait in agroforestry, such as fruit, wood, or biomass production, results in more and more researchers interested in adopting the CRISPR-Cas system for their investigations in woody plants. To achieve this purpose, we provide a step-by-step guide considering all critical decisions that a researcher must consider. Eliminating the transgenic scarfs of targeted genome editing, i.e., the Cas DNA sequence and other transgenic sequences within the genome after plant regeneration, is particularly difficult for perennial plants, where segregation by sexual seed production takes an extremely long period. In this chapter, we also summarize the potential strategies to face the challenge of applying next-generation plant breeding to create commercial genotypes with high ecological and economic value in trees.