Overcoming diverse homologous recombinations and single chimeric guide RNA competitive inhibition enhances Cas9-based cyclical multiple genes coediting in filamentous fungi
Overcoming diverse homologous recombinations and single chimeric guide RNA competitive inhibition enhances Cas9-based cyclical multiple genes coediting in filamentous fungi
复制标题
克服多种同源重组和单一嵌合指导RNA竞争性抑制增强丝状真菌中基于Cas9的周期性多基因共编辑
DOI：
10.1111/1462-2920.15477
复制
发表时间：
2021-05-06
影响因子：
5.1
通讯作者：
Zhang, Ruifu
中科院分区：
文献类型：
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作者：
Miao, Youzhi;Xia, Yanwei;Zhang, Ruifu
破解丝状真菌复杂的细胞行为和推进丝状真菌的生物技术应用增加了对大量靶基因进行遗传操作的需求。目前的策略不能同时循环共编辑多个基因。在这项研究中，我们首先揭示了丝状真菌无标记编辑中存在多种同源重组（HR）类型，然后证明sgRNA效率介导的竞争性抑制导致多个基因位点在共编辑过程中的低整合，这是限制多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，以大大增强所需的HR类型，并对多个供体DNA应用了一种新的选择标记标记策略，其中仅标记了具有最低sgRNA效率的供体DNA。结合这些策略，我们成功地开发了一种方便的方法，在不同的丝状真菌中循环共编辑多个基因。此外，不同的HR导致了一个有用的和方便的一步的方法结合基因破坏和互补的基因功能研究。该研究为丝状真菌基因功能研究提供了一种一步到位的方法，也为丝状真菌多基因的循环共编辑提供了一种有效的策略。
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.