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