More and more available fungal genome sequence data reveals a large number of secondary metabolite (SM) biosynthetic "dark matter" to be discovered. Heterogeneous expression approach is one of the most effective way to exploit these novel natural products, but it is limited by cloning of entire biosynthetic gene clusters (BGCs) without errors. So far, few effective technologies were developed to manipulate the specific large DNA fragments in the filamentous fungi. Here, we developed a fungal SM gene clusters capturing system based on CRISPR/Cas9 cleavage in vitro. In this system, Cas9 protein was purified and CRISPR guide sequences in combination with in vivo yeast recombination were rationally designed. By the targeted cleavages of plasmid DNAs with linear (8.5 kb) or circular (8.5 kb and 28 kb) states, these plasmids were cleaved precisely indicating the high efficiency of this system. Furthermore, we successfully captured the entire Nrc gene cluster from the genomic DNA of Neosartorya fischeri. Our results provide an easy and efficient approach to manipulate fungal genomic DNA based on the in vitro application of Cas9 endonuclease. Our methodology will lay the foundation for capturing the entire BGCs in filamentous fungi and accelerate the fungal SM mining.

中文翻译：

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用于捕获真菌次级代谢产物基因簇的 CRISPR/Cas9 切割系统。

越来越多可用的真菌基因组序列数据揭示了大量次级代谢产物（SM）生物合成“暗物质”有待发现。异源表达方法是利用这些新型天然产物的最有效方法之一，但它受到无错误克隆整个生物合成基因簇 (BGC) 的限制。到目前为止，几乎没有开发出有效的技术来操纵丝状真菌中特定的大 DNA 片段。在这里，我们开发了一种基于 CRISPR/Cas9体外切割的真菌 SM 基因簇捕获系统。在该系统中，纯化Cas9蛋白并结合体内的CRISPR指导序列合理设计酵母重组。通过对具有线性 (8.5 kb) 或环状 (8.5 kb 和 28 kb) 状态的质粒 DNA 进行靶向切割，这些质粒被精确切割，表明该系统的高效性。此外，我们成功地从Neosartorya fischeri的基因组 DNA 中捕获了整个Nrc基因簇。我们的结果提供了一种简单有效的方法来基于Cas9 核酸内切酶的体外应用来操作真菌基因组 DNA。我们的方法将为捕获丝状真菌中的整个 BGC 并加速真菌 SM 挖掘奠定基础。