CRISPR/Cas9介导的基因组定点编辑技术在丝状真菌中的研究进展

Research progress of CRISPR/Cas9 system in genome targeted editing of filamentous fungi

CRISPR/Cas9技术是在特定的RNA引导下,利用特异的核酸酶实现对基因组进行编辑的新技术。自2013年该技术体系建立起来已成功应用于动物、植物及真菌中。本文简述了3种基于核酸酶的基因编辑技术及其应用,概述了CRISPR/Cas9系统的组成及其作用机理,总结了CRISPR/Cas9在模式真菌酿酒酵母及丝状真菌中的应用,并就在丝状真菌中应用该技术时sg RNA表达盒的设计、Cas9表达盒的优化、抗性标记的筛选、受体的选择等方面提出具体的研究方法。另外,针对该技术应用过程中出现的脱靶效应、Cas9核定位信号的添加、启动子的选择及多个靶基因的编辑等问题提出了建议与展望,希望能够为初次涉足该领域的科研人员提供理论参考和技术支持。

CRISPR（clustered regularly interspaced short palindromic repeats）/Cas9 technology, established in 2013, guided by special RNA and initiated by endonuclease, has been developed into the third generation of genome editing technology, which is widely used in genome editing among varieties of species including animals, plants, bacteria and fungi with high efficiency and specificity. This review briefly introduces the characteristics and the application of three genome editing technologies based on endonucleases, describes the composes and mechanism of CRISPR/Cas9 mediated genome editing, summarizes the application of this tool in Saccharomyces cerevisiae and filamentous fungi in genome editing. In the other hand, we put forward the protocol about using CRISPR/Cas9 technology in filamentous fungi, such as the design of sg RNA cassette, the optimization of Cas9 cassette, the screening of resistance marker, the selection of receptor, and so on. Furthermore, some suggestions were given about the problems which often encounter in practical application, such as off-target effects, the addition of Cas9 nuclear localization signal, the selection of promoter, and multi-gene editing, to provide a theoretical and technological references for the beginners in filamentous fungus gene editing field.