Establishment of a Novel CRISPR/Cas9-Based Multiplex Editing System in the Citrus Postharvest Pathogen Penicillium digitatum

Penicillium digitatum, the causal fungus of the citrus green mold, leads to substantial postharvest losses in the citrus industry. In this study, we engineered a versatile CRISPR/Cas9-mediated gene editing platform capable of generating multiple sgRNAs from a single polycistronic transcript. By incorporating endogenous tRNAs and the strong promoter PdMLE1 into the CRISPR/Cas9 system, the efficiency of single gene editing can reach up to 94.2%. By taking advantage of the efficient shearing and processing capabilities of tRNA, the platform enabled multiplex editing with efficiencies of 44.4% for two-gene and 33.3% for three-gene modifications, respectively. After two rounds of three-gene editing, we were able to successfully obtain hextuple-gene mutants. Finally, functional characterization revealed that the target polysaccharide-lyase-encoding genes play limited roles in pathogenicity in P. digitatum . Taken together, our results represent a powerful tool for genome engineering in P. digitatum, facilitating research into its pathogenesis.