Virus‐Induced Genome Editing in the Parasitic Plant Phtheirospermum japonicum

Summary Statement Phtheirospermum japonicum is a genetic model for parasitic Orobanchaceae, a family that includes agriculturally important weeds such as Striga , Orobanche , and Phelipanche . Functional genetic studies in these species are limited by the lack of stable transgenesis protocols, restricting heritable CRISPR/Cas9 genome editing. This challenge is common to many non‐standard model plants and crops. Here, we combined Agrobacterium rhizogenes ‐mediated transformation with virus‐assisted delivery of the ultracompact TnpB nuclease to achieve stable, transgene‐free, heritable genome editing in P. japonicum , independent of tissue regeneration or floral dip transformation. Homozygous knockouts were obtained within one to two generations. Modifications to this system increased editing efficiency and expanded the target space for editing. Our approach enables targeted functional genetic studies in parasitic plants and offers potential for broad application in non‐classical model species and crops.