SAM-Targeted CRISPR-Cas9 RNP Delivery Combined with Leaf Regeneration Enables DNA-Free, Non-Chimeric Genome Editing in ‘Fuji’ Apple

DNA-free genome editing is a promising strategy for the genetic improvement of horticultural crops and fruit trees because it enables targeted mutagenesis without stable genetic transformation. In planta particle bombardment (iPB) delivers CRISPR-Cas9 ribonucleoproteins (RNPs) directly into shoot apical meristems (SAMs), enabling heritable genome editing without the use of tissue culture-based transformation systems. However, the practical application of iPB-mediated editing in fruit trees is limited by the frequent occurrence of chimerism, which cannot be readily eliminated through sexual segregation while maintaining the genetic background of elite cultivars. To overcome this limitation, we combined iPB-mediated RNP delivery with regeneration from edited leaf tissues (iPB-REG). Using this approach, we targeted the self-incompatibility gene S9-RNase in the elite apple cultivar ‘Fuji’ and efficiently recovered non-chimeric edited plants. These results establish iPB-REG as a practical strategy for producing uniform genome-edited fruit trees and provide a valuable platform for DNA-free genetic improvement and functional genomics in clonally propagated perennial crops.