Marker-free genome editing in the edible mushroom, Pleurotus ostreatus, using transient expression of genes required for CRISPR/Cas9 and for selection

Abstract In a previous study, we reported a transient transformation system using repeated screening for hygromycin B (Hyg) resistance in the basidiomycete Ceriporiopsis subvermispora . In the present study, by combining this technique with CRISPR/Cas9, we demonstrated successful marker-free genome editing in Pleurotus ostreatus , which is one of the most economically important cultivated mushrooms as well as a model white-rot fungus. At first, transformant selection mediated by the transient expression of marker genes was demonstrated using a plasmid harboring the Hyg resistance gene ( hph ) in P. ostreatus . Then, genome editing of fcy1 , which confers 5-fluorocytosine (5-FC) resistance to the host cell, was performed by the transient expression of Cas9, gRNA, and hph and strains with 5-FC resistance and Hyg sensitivity were isolated. Additionally, genome editing of fcy1 in these strains was confirmed by Sanger sequencing. To our knowledge, this is the first report of marker-free genome editing through the transient expression of Cas9, gRNA, and hph in agaricomycetes, which opens the door for repeated genome editing in these fungi.