Marker-free genome editing in the edible mushroom, Pleurotus ostreatus, using transient expression of genes required for CRISPR/Cas9 and for selection
Marker-free genome editing in the edible mushroom, Pleurotus ostreatus, using transient expression of genes required for CRISPR/Cas9 and for selection
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DOI：
10.1186/s10086-022-02033-6
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发表时间：
2022-12-01
影响因子：
2.9
通讯作者：
Honda, Yoichi
中科院分区：
文献类型：
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作者：
Koshi, Daishiro;Ueshima, Hiroki;Honda, Yoichi
在之前的研究中，我们报道了一种瞬时转化系统，该系统使用反复筛选颠覆性宫颈担子菌对潮霉素B （Hyg）的抗性。在本研究中，我们将该技术与CRISPR/Cas9技术相结合，成功地对平菇（Pleurotus ostreatus）进行了无标记基因组编辑。平菇是一种经济上最重要的栽培蘑菇，也是一种典型的白腐菌。首先，利用含Hyg抗性基因（hph）的质粒，证明了标记基因瞬时表达介导的转化选择。然后，通过瞬时表达Cas9、gRNA和hph对宿主细胞具有5-氟胞嘧啶（5-FC）抗性的fcy1进行基因组编辑，分离出具有5-FC抗性和Hyg敏感性的菌株。此外，Sanger测序证实了这些菌株中fcy1的基因组编辑。据我们所知，这是第一个通过在真菌中瞬时表达Cas9、gRNA和hph进行无标记基因组编辑的报道，这为在这些真菌中进行重复基因组编辑打开了大门。
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.