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

Autor: Daishiro Koshi, Hiroki Ueshima, Moriyuki Kawauchi, Takehito Nakazawa, Masahiro Sakamoto, Mana Hirata, Kosuke Izumitsu, Takuya Sumita, Toshikazu Irie, Yoichi Honda
Jazyk: angličtina
Rok vydání: 2022
Předmět:
Zdroj: Journal of Wood Science, Vol 68, Iss 1, Pp 1-8 (2022)
Druh dokumentu: article
ISSN: 1435-0211
1611-4663
DOI: 10.1186/s10086-022-02033-6
Popis: 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.
Databáze: Directory of Open Access Journals
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