ABSTRACT

Until recently, classical breeding has been used to generate improved commercial mushroom strains; however, classical breeding remains to be laborious and time-consuming. In this study, we performed gene mutagenesis using Cas9 ribonucleoprotein (Cas9 RNP) as a plasmid-free genome editing in Pleurotus ostreatus, which is one of the most economically important cultivated mushrooms. The pre-assembled Cas9/sgRNA targeting pyrG was introduced into protoplasts of a wild-type monokaryotic P. ostreatus strain PC9, which resulted in a generation of strains exhibiting resistance to 5-fluoroorotic acid. Small insertions/deletions at the target site were identified using genomic PCR followed by sequencing. The results showed Cas9 RNP-assisted gene mutagenesis could be applied for the molecular breeding in P. ostreatus and in other edible mushroom strains. Furthermore, gene disruption via split-marker recombination using the Cas9 RNP system was also successfully demonstrated in wild-type P. ostreatus PC9. This method could overcome the disadvantages of NHEJ-deficiency in conventional studies with gene targeting, and also difficulty in gene targeting in various non-model agaricomycetes.

中文翻译：

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在平菇中使用预组装的 Cas9 核糖核蛋白和分裂标记重组进行基因打靶

摘要

直到最近，经典育种已被用于产生改良的商业蘑菇品系。然而，经典育种仍然费时费力。在本研究中，我们使用 Cas9 核糖核蛋白 (Cas9 RNP) 作为无质粒基因组编辑平菇（Pleurotus ostreatus）进行基因诱变，平菇是经济上最重要的栽培蘑菇之一。将靶向pyrG的预组装 Cas9/sgRNA引入野生型单核P. ostreatus 的原生质体中菌株 PC9，这导致一代对 5-氟乳清酸表现出抗性的菌株。使用基因组 PCR 和测序鉴定目标位点的小插入/缺失。结果表明Cas9 RNP辅助基因诱变可用于P. ostreatus和其他食用菌菌株的分子育种。此外，在野生型P. ostreatus PC9 中也成功证明了使用 Cas9 RNP 系统通过分裂标记重组进行基因破坏。该方法可以克服传统基因打靶研究中NHEJ缺陷的缺点，以及各种非模型伞形孢菌的基因打靶困难。