The establishment of multiple knockout mutants of Colletotrichum orbiculare by CRISPR-Cas9 and Cre-loxP systems
The establishment of multiple knockout mutants of Colletotrichum orbiculare by CRISPR-Cas9 and Cre-loxP systems
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DOI：
10.1016/j.fgb.2023.103777
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发表时间：
2023-02-01
影响因子：
3
通讯作者：
Takano, Yoshitaka
中科院分区：
文献类型：
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作者：
Yamada, Kohji;Yamamoto, Toya;Takano, Yoshitaka
以炭疽菌为模式真菌，研究植物-真菌相互作用的分子机制。虽然通过同源重组（HR）的基因中断建立了C。但是，这种方法由于其低效率而费力。在这里，我们开发了产生多个敲除C突变体的方法。有效地圆。我们首先发现CRISPR-Cas9系统大大提高了基因靶向效率。通过瞬时引入CRISPR-Cas9载体，超过90%的获得的转化体是敲除突变体。此外，我们优化了一个自切除Cre loxP标记回收系统。因为所需选择性标记的有限可用性阻碍了连续的基因破坏。在该系统中，整合的选择性标记可通过由木糖诱导型启动子驱动的Cre重组酶从基因组中去除，使得相同的选择性标记能够重复用于下一次转化。使用我们的CRISPR-Cas9和Cre-loxP系统，我们试图鉴定参与真菌毒力的功能性糖转运蛋白。假定的奎尼酸转运蛋白基因的多重破坏限制了真菌在含有奎尼酸作为唯一碳源的培养基上的生长，证实了它们作为奎尼酸转运蛋白的功能。然而，我们的分析表明，奎尼酸的收购是有害的感染宿主植物。此外，我们成功地在菌株中构建了17个纤维二糖转运蛋白基因的突变。从我们在本研究中建立的敲除突变体的数据中，我们推断使用CRISPR-Cas9和Cre-loxP系统进行的重复基因破坏不会对真菌毒力和生长产生不利影响。因此，这些系统将是强大的工具，执行一个系统的功能丧失的方法为C。圆形的
Colletotrichum orbiculare is employed as a model fungus to analyze molecular aspects of plant-fungus interactions. Although gene disruption via homologous recombination (HR) was established for C. orbiculare, this approach is laborious due to its low efficiency. Here we developed methods to generate multiple knockout mutants of C. orbiculare efficiently. We first found that CRISPR-Cas9 system massively promoted gene-targeting efficiency. By transiently introducing a CRISPR-Cas9 vector, more than 90% of obtained transformants were knockout mutants. Furthermore, we optimized a self-excision Cre-loxP marker recycling system for C. orbiculare because a limited availability of desired selective markers hampers sequential gene disruption. In this system, the integrated selective marker is removable from the genome via Cre recombinase driven by a xylose-inducible promoter, enabling the reuse of the same selective marker for the next transformation. Using our CRISPR-Cas9 and Cre-loxP systems, we attempted to identify functional sugar transporters involved in fungal virulence. Multiple disruptions of putative quinate transporter genes restricted fungal growth on media containing quinate as a sole carbon source, confirming their functionality as quinate transporters. However, our analyses showed that quinate acquisition was dispensable for infection to host plants. In addition, we successfully built mutations of 17 cellobiose transporter genes in a strain. From the data of knockout mutants that we established in this study, we inferred that repetitive rounds of gene disruption using CRISPR-Cas9 and Cre-loxP systems do not cause adverse effects on fungal virulence and growth. Therefore, these systems will be powerful tools to perform a systematic loss-of-function approach for C. orbiculare.