State Key Laboratory of Mycology and CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, P.R. China.
Savaid Medical School, University of Chinese Academy of Sciences, Beijing 100049, P.R. China.
J Microbiol Biotechnol. 2021 Jan 28;31(1):8-15. doi: 10.4014/jmb.2008.08040.
More and more available fungal genome sequence data reveal a large amount of secondary metabolite (SM) biosynthetic 'dark matter' to be discovered. Heterogeneous expression is one of the most effective approaches to exploit these novel natural products, but it is limited by having to clone entire biosynthetic gene clusters (BGCs) without errors. So far, few effective technologies have been developed to manipulate the specific large DNA fragments in filamentous fungi. Here, we developed a fungal BGC-capturing system based on CRISPR/Cas9 cleavage in vitro. In our system, Cas9 protein was purified and CRISPR guide sequences in combination with in vivo yeast assembly were rationally designed. Using targeted cleavages of plasmid DNAs with linear (8.5 kb) or circular (8.5 kb and 28 kb) states, we were able to cleave the plasmids precisely, demonstrating the high efficiency of this system. Furthermore, we successfully captured the entire gene cluster from the genomic DNA of . 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 a foundation for capturing entire groups of BGCs in filamentous fungi and accelerate fungal SMs mining.
越来越多可用的真菌基因组序列数据揭示出大量有待发现的次级代谢产物（SM）生物合成“暗物质”。异质表达是开发这些新型天然产物的最有效方法之一，但它受到必须克隆整个生物合成基因簇（BGC）而不出现错误的限制。到目前为止，很少有有效的技术能够用于操纵丝状真菌中的特定大片段 DNA。在这里，我们开发了一种基于 CRISPR/Cas9 在体外切割的真菌 BGC 捕获系统。在我们的系统中，纯化了 Cas9 蛋白，并合理设计了 CRISPR 指导序列与体内酵母组装相结合。使用线性（8.5 kb）或圆形（8.5 kb 和 28 kb）状态的质粒 DNA 的靶向切割，我们能够精确地切割质粒，证明了该系统的高效率。此外，我们还成功地从. 的基因组 DNA 中捕获了整个基因簇。我们的结果提供了一种基于 Cas9 内切酶体外应用的简便、高效的方法来操纵真菌基因组 DNA。我们的方法将为在丝状真菌中捕获整个 BGC 组群并加速真菌 SM 挖掘奠定基础。
