PDF(Pubmed)
Abstract：
Reverse genetic approaches are common tools in genomics for elucidating gene functions, involving techniques such as gene deletion followed by screening for aberrant phenotypes. If the generation of gene deletion mutants fails, the question arises whether the failure stems from technical issues or because the gene of interest (GOI) is essential, meaning that the deletion causes lethality. In this report, we introduce a novel method for assessing gene essentiality using the phytopathogenic ascomycete Magnaporthe oryzae. The method is based on the observation that telomere vectors are lost in transformants during cultivation without selection pressure. We tested the hypothesis that essential genes can be identified in deletion mutants co-transformed with a telomere vector. The M. oryzae gene MoPKC, described in literature as essential, was chosen as GOI. Using CRISPR/Cas9 technology transformants with deleted GOI were generated and backed up by a telomere vector carrying a copy of the GOI and conferring fenhexamid resistance. Transformants in which the GOI deletion in the genome was not successful lost the telomere vector on media without fenhexamid. In contrast, transformants with confirmed GOI deletion retained the telomere vector even in absence of fenhexamid selection. In the latter case, the maintenance of the telomere indicates that the GOI is essential for the surveillance of the fungi, as it would have been lost otherwise. The method presented here allows to test for essentiality of genes when no mutants can be obtained from gene deletion approaches, thereby expanding the toolbox for studying gene function in ascomycetes.
摘要：
反向遗传学方法是基因组学中阐明基因功能的常用工具，涉及基因缺失等技术，然后筛选异常表型。如果基因缺失突变体的产生失败，问题是失败是源于技术问题还是因为感兴趣的基因（GOI）是必不可少的，这意味着删除会导致致命。在这份报告中,我们介绍了一种使用植物病原性子囊菌稻瘟病菌评估基因重要性的新方法。该方法基于在没有选择压力的培养过程中端粒载体在转化体中丢失的观察。我们测试了以下假设：可以在与端粒载体共转化的缺失突变体中鉴定必需基因。米曲霉基因MoPKC，在文献中被描述为必不可少的，被选为GOI。使用CRISPR/Cas9技术,产生具有删除的GOI的转化体,并由携带GOI拷贝并赋予fenhexamid抗性的端粒载体支持。基因组中GOI缺失未成功的转化体在没有fenhexamid的培养基上丢失了端粒载体。相比之下,即使没有fenhexamid选择，已确认GOI缺失的转化体仍保留端粒载体。在后一种情况下，端粒的维持表明GOI对真菌的监测至关重要，否则它就会丢失。当无法从基因缺失方法中获得突变体时，此处介绍的方法可以测试基因的重要性。从而扩大了子囊菌基因功能研究的工具箱。
