Jiangsu Provincial Key Lab for Organic Solid Waste Utilization, National Engineering Research Center for Organic-based Fertilizers, Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization, Nanjing Agricultural University, Nanjing, 210095, China.
Key Laboratory of Microbial Resources Collection and Preservation, Ministry of Agriculture, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
Environ Microbiol. 2021 Jun;23(6):2937-2954. doi: 10.1111/1462-2920.15477. Epub 2021 May 6.
Deciphering the complex cellular behaviours and advancing the biotechnology applications of filamentous fungi increase the requirement for genetically manipulating a large number of target genes. The current strategies cannot cyclically coedit multiple genes simultaneously. In this study, we firstly revealed the existence of diverse homologous recombination (HR) types in marker-free editing of filamentous fungi, and then, demonstrated that sgRNA efficiency-mediated competitive inhibition resulted in the low integration of multiple genetic sites during coediting, which are the two major obstacles to limit the efficiency of cyclically coediting of multiple genes. To overcome these obstacles, we developed a biased cutting strategy by Cas9 to greatly enhance the desired HR type and applied a new selection marker labelling strategy for multiple donor DNAs, in which only the donor DNA with the lowest sgRNA efficiency was labelled. Combined with these strategies, we successfully developed a convenient method for cyclically coediting multiple genes in different filamentous fungi. In addition, diverse HRs resulted in a useful and convenient one-step approach for gene functional study combining both gene disruption and complementation. This research provided both a useful one-step approach for gene functional study and an efficient strategy for cyclically coediting multiple genes in filamentous fungi.
解析丝状真菌复杂的细胞行为并推进生物技术应用，这增加了对大量目标基因进行遗传操作的需求。目前的策略无法循环地同时编辑多个基因。在本研究中，我们首先揭示了无标记编辑丝状真菌中存在多种同源重组（HR）类型，然后证明 sgRNA 效率介导的竞争抑制导致在共编辑过程中多个遗传位点的低整合，这是限制多个基因循环共编辑效率的两个主要障碍。为了克服这些障碍，我们通过 Cas9 开发了一种偏向切割策略，从而大大增强了所需的 HR 类型，并应用了一种新的用于多个供体 DNA 的选择标记标记策略，其中只有 sgRNA 效率最低的供体 DNA 被标记。结合这些策略，我们成功开发了一种在不同丝状真菌中循环共编辑多个基因的简便方法。此外，不同的 HR 导致了一种有用且方便的一步法，可用于结合基因缺失和互补进行基因功能研究。这项研究为基因功能研究提供了一种有用的一步法，以及在丝状真菌中循环共编辑多个基因的有效策略。
