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- Title
Marker-free genome editing in the edible mushroom, Pleurotus ostreatus, using transient expression of genes required for CRISPR/Cas9 and for selection.
- Authors
Koshi, Daishiro; Ueshima, Hiroki; Kawauchi, Moriyuki; Nakazawa, Takehito; Sakamoto, Masahiro; Hirata, Mana; Izumitsu, Kosuke; Sumita, Takuya; Irie, Toshikazu; Honda, Yoichi
- 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.
- Subjects
PLEUROTUS ostreatus; EDIBLE mushrooms; GENE expression; CRISPRS; CULTIVATED mushroom; GENOME editing; DISEASE resistance of plants
- Publication
Journal of Wood Science, 2022, Vol 68, Issue 1, p1
- ISSN
1435-0211
- Publication type
Article
- DOI
10.1186/s10086-022-02033-6