期刊
JOURNAL OF WOOD SCIENCE
卷 68, 期 1, 页码 -出版社
SPRINGER JAPAN KK
DOI: 10.1186/s10086-022-02033-6
关键词
Transient transformation; Genome editing; Agaricomycete; Mushroom; Wood-rot fungi; Pleurotus ostreatus; CRISPR/Cas9
资金
- KAKENHI [18H02254, 18KK0178, 19K22332, 21K18224]
- JSPS Bilateral Program [JPJSBP 120208402, 120209920]
- Kyoto University Foundation
- Grants-in-Aid for Scientific Research [21K18224, 19K22332, 18H02254, 18KK0178] Funding Source: KAKEN
In this study, marker-free genome editing was achieved in Pleurotus ostreatus by combining CRISPR/Cas9 with a transient transformation system. This opens up possibilities for repeated genome editing in these fungi.
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.
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