Abstract
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.
Highlights
Mushroom-forming basidiomycetes are unique in that they develop a three-dimensional organized fruiting body useful for the production of a wide range of secondary metabolites as well as supplying food globally
The most widespread genome editing technology is the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) system, which consists of two components: Cas[9] nuclease, and a guide RNA that targets the genome sequence of interest[13]
The present study demonstrated CRISPR/Cas9-based genome editing and high-throughput transformation methods in the mushroom-forming basidiomycete C. cinerea
Summary
Mushroom-forming basidiomycetes are unique in that they develop a three-dimensional organized fruiting body useful for the production of a wide range of secondary metabolites as well as supplying food globally. In C. cinerea, gene targeting methods for application to genetic studies have been developed using Δku[70] or Δlig[4] mutants[6]; high-throughput reverse genetics and appropriate reporter assays remain to be established[7]. The present study demonstrated CRISPR/Cas9-based genome editing and high-throughput transformation methods in the mushroom-forming basidiomycete C. cinerea. CRISPR/Cas[9] customization to C. cinerea was achieved via identification of a novel, constitutively active, promoter screened from a high-throughput transformation method using cryopreserved protoplasts. We developed a novel luciferase assay that takes only 1 day to measure promoter activities in C. cinerea protoplasts This high-throughput transformation and the CRISPR/ Cas[9] system customized for mushroom-forming basidiomycetes could contribute to the acceleration of genetic studies and molecular breeding in these fungi
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