Efficient CRISPR-Cas9 delivery and transgene-free multiplex genome editing in plants using cymbidium mosaic virus-derived vectors.

Virus-induced genome editing (VIGE) has become a useful method by enabling transient delivery of gene-editing reagents; however, many viral systems face limitations in cargo size, host range, or reliance on transgenic Cas9-expressing plants. In this study, we developed a cymbidium mosaic virus (CymMV)-based VIGE platform that enables simultaneous expression of Streptococcus pyogenes Cas9 (SpCas9) and one or more guide RNAs (gRNAs) from a single viral RNA. In Nicotiana benthamiana, this system induced editing in the Phytoene desaturase (PDS) gene, with indel rates exceeding 50% within 6 days after inoculation, outperforming traditional delivery methods by about fivefold. Notably, over 80% of regenerated plants contained targeted mutations, and 82% of these were both transgene- and virus-free, including tetra-allelic knockouts directly in the M0 generation. Adding a Ruby-based visual counterselection marker enabled rapid, reliable identification of transgene-free, edited plants without antibiotic selection. When adapted to Phalaenopsis aphrodite orchids, the platform efficiently edited the PaPDS gene, achieving a 47% indel frequency at 20 days post-inoculation, with visible bleaching in leaf tissue from inoculated protocorm-like bodies. Additionally, expressing multiple gRNAs from a single CymMV replicon enabled multiplex editing in orchid tissues, demonstrating the system's versatility for complex, polyploid crops. Our findings broaden the use of VIGE in orchids and provide a reliable framework for precision plant breeding.