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 (Imai et al., 2020; Kumagai et al., 2022). Because SAMs contain subepidermal (L2) cells that give rise to germ cells, the successful delivery of Cas9 RNPs into these cells can generate heritable mutations (Kumagai et al., 2022). However, a major limitation of iPB-mediated editing is chimerism. Although chimerism can be eliminated through genetic segregation in annual crops, this strategy is impractical in fruit trees because sexual reproduction disrupts the highly heterozygous genetic backgrounds of elite cultivars. Therefore, methods capable of eliminating chimerism while preserving cultivar identity are required. Here, iPB-mediated RNP delivery was combined with regeneration from leaf tissue (iPB-REG), enabling the efficient recovery of chimera-free edited plants in the elite apple cultivar 'Fuji'.