In vitro CRISPR/Cas9-RNP cleavage of CcYUC1 in Coffea canephora.

An in vitro CRISPR/Cas9-RNP system efficiently cleaves CcYUC1 in Coffea canephora, establishing a foundation for DNA-free genome editing in coffee. Somatic embryogenesis depends on auxin biosynthesis and signaling; however, functional validation of candidate genes remains limited. In this study, we identified CcYUC1, a putative flavin monooxygenase gene associated with indole-3-acetic acid biosynthesis, during SE induction in Coffea canephora. CcYUC1 transcripts accumulated during the early stages of SE, suggesting a role in embryogenic induction. To establish a genome editing platform in coffee, we designed a CRISPR/Cas9 ribonucleoprotein (RNP) system targeting exon 4 of CcYUC1. In vitro cleavage assays confirmed specific and efficient digestion of the target, achieving over 80% cleavage under optimized Cas9/sgRNA conditions. These findings establish a proof-of-concept CRISPR/Cas9-RNP platform for DNA-free genome editing in coffee and provide a basis for future functional studies of auxin biosynthesis during somatic embryogenesis.