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

<i>Penicillium digitatum</i>, 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 <i>P. digitatum</i>. Taken together, our results represent a powerful tool for genome engineering in <i>P. digitatum</i>, facilitating research into its pathogenesis.