Suzuki, Hiroko Tokushima University
下北, 英輔 Tokushima University|Tokushima Prefectural Agriculture, Forestry and Fisheries Technology Support Center
Chiba, Hirofumi Tokushima University
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, CcDED1pro, 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 CcDED1pro 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.
Supplementary tables and figures : srep_7_1260_s1.pdf
© The Author(s) 2017
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srep_7_1260.pdf 5.31 MB
srep_7_1260_s1.pdf 2.87 MB