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Mushroom-forming basidiomycetes produce a wide range of metabolites and have great value not only as food but also as an important global natural resource. Here, we demonstrate CRISPR/Cas9-based genome editing in the model species  Coprinopsis cinerea . Using a high-throughput reporter assay with cryopreserved protoplasts, we identified a novel promoter,  CcDED1    pro  , with seven times stronger activity in this assay than the conventional promoter  GPD2 . To develop highly efficient genome editing using CRISPR/Cas9 in  C. cinerea , we used the  CcDED1    pro   to express Cas9 and a  U6-snRNA  promoter from  C .  cinerea  to express  gRNA . Finally, CRISPR/Cas9-mediated  GFP  mutagenesis was performed in a stable  GFP  expression line. Individual genome-edited lines were isolated, and loss of GFP function was detected in hyphae and fruiting body primordia. This novel method of high-throughput CRISPR/Cas9-based genome editing using cryopreserved protoplasts should be a powerful tool in the study of edible mushrooms.

Genome editing in the mushroom-forming basidiomycete Coprinopsis cinerea, optimized by a high-throughput transformation system