Establishment of a CRISPR/Cas9-Mediated Genome Editing System in Physalis grisea by Targeting the PgPDS Gene

Physalis grisea is an orphan crop with significant economic and medicinal potential. Although initial genome editing applications have recently emerged for Physalis species, the development and optimization of highly efficient, visually traceable Agrobacterium-mediated editing platforms remain crucial for advancing its functional genomics. This study uses the phytoene desaturase (PDS) gene—a key enzyme in the carotenoid biosynthetic pathway—as a visual reporter to develop a CRISPR/Cas9-mediated genome editing platform in P. grisea. A dual-target guide RNA (sgRNA) expression vector was constructed, and transgenic plants were successfully generated via Agrobacterium-mediated transformation of hypocotyl explants. Strikingly, phenotypic observations revealed that the regenerated mutants exhibited characteristic complete albino or green-white chimeric phenotypes, accompanied by distinct developmental retardation and dwarfing. Physiological quantitative analysis showed that total chlorophyll and carotenoid contents in the mutant leaves were significantly reduced by over 70% and 78%, respectively. Targeted sequencing further confirmed that the CRISPR/Cas9 system efficiently induced various mutations at the PgPDS locus (derived from Physalis grisea)—including fragment deletions, 1–4 bp insertions, and 2–3 bp substitutions—revealing a specific preference for non-homologous end joining (NHEJ) repair. In summary, this study not only validates the suitability of PgPDS as a reporter gene but also successfully establishes a robust genome editing technical system for P. grisea, providing a solid foundation for future functional genomics research and molecular breeding in this crop.