CRISPR/Cas9-mediated efficient genome editing via blastospore-based transformation in entomopathogenic fungus Beauveria bassiana.
CRISPR/Cas9-mediated efficient genome editing via blastospore-based transformation in entomopathogenic fungus Beauveria bassiana.
复制标题
CRISPR/Cas9介导的昆虫病原真菌白僵菌中基于芽生孢子的高效基因组编辑
DOI：
10.1038/srep45763
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发表时间：
2017-04-03
影响因子：
4.6
通讯作者：
Wang S
中科院分区：
文献类型：
--
作者：
Chen J;Lai Y;Wang L;Zhai S;Zou G;Zhou Z;Cui C;Wang S
球孢白僵菌是一种环境友好的替代化学杀虫剂对各种农业害虫和人类疾病的载体。然而，由于其对非生物胁迫的敏感性和缓慢杀灭，其应用受到限制。了解其分子致病机理和生理特性将有助于提高真菌的生产性能。功能丧失突变是表征基因功能的最有力的工具，但它受到同源重组率低和选择标记可用性有限的阻碍。在这里，通过将尿苷营养缺陷型作为受体和供体DNA，携带营养缺陷型互补基因ura 5作为选择标记，与基于芽生孢子的转化系统相结合，我们在B中建立了高效、低假阳性背景和具有成本效益的CRISPR/Cas9介导的基因编辑系统。巴西纳。该系统已被证明是一个简单而强大的工具，靶向基因敲除和/或敲入B。在一个单一的基因中断。我们进一步证明了我们的系统允许在单个转化中通过同源定向修复同时破坏多个基因。这项技术将使我们能够研究功能冗余基因，并具有极大的潜力，大大加快B的功能基因组学研究。巴西纳。
Beauveria bassiana is an environmentally friendly alternative to chemical insecticides against various agricultural insect pests and vectors of human diseases. However, its application has been limited due to slow kill and sensitivity to abiotic stresses. Understanding of the molecular pathogenesis and physiological characteristics would facilitate improvement of the fungal performance. Loss-of-function mutagenesis is the most powerful tool to characterize gene functions, but it is hampered by the low rate of homologous recombination and the limited availability of selectable markers. Here, by combining the use of uridine auxotrophy as recipient and donor DNAs harboring auxotrophic complementation gene ura5 as a selectable marker with the blastospore-based transformation system, we established a highly efficient, low false-positive background and cost-effective CRISPR/Cas9-mediated gene editing system in B. bassiana. This system has been demonstrated as a simple and powerful tool for targeted gene knock-out and/or knock-in in B. bassiana in a single gene disruption. We further demonstrated that our system allows simultaneous disruption of multiple genes via homology-directed repair in a single transformation. This technology will allow us to study functionally redundant genes and holds significant potential to greatly accelerate functional genomics studies of B. bassiana.