BACKGROUND Trichophyton rubrum is an obligate human parasitic fungus and responsible for approximately 80-90% of dermatomycosis in human. Molecular genetic manipulations of this pathogen are challenging and available tools and protocols are only rudimentary. We adapt molecular genetics methods of well established fungal model organism, to knock out genes in T. rubrum. For the adaptation, crucial modifications are necessary. With the implementation of in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, it is possible to adapt molecular genetic methods, to knock out genes in T. rubrum. RESULTS The gene knock-out method is based on integration of a selection marker into the target site, to interrupt the gene translation. The target gene gets preassigned by the homologous sequence of the in vitro synthesized Cas9-sgRNA ribonucleoprotein complex. To develop the method, we first isolated and characterized a T. rubrum strain with a high amount of microconidia. Next, we developed a transformation protocol, whereby the Cas9-sgRNA ribonucleoprotein gets delivered into the fungal protoplast by the PEG method. We knocked out the URA3 gene and resulted, as predicted, uracil auxotrophic strains. These strains can be used for specific gene knock-outs by reintegrating the URA3 fragment and selection on uracil lacking cultivation media. Exemplary, we knocked out the TRP3 gene and got the predicted phenotype, tryptophan auxotrophic strains. The mutation had been verified by sequencing. CONCLUSIONS We developed a method, based on in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, for target specific gene knock-outs in T. rubrum. We knocked out the Ura3 gene and resulted uracil auxotrophic strains. These strains were used for target specific gene knock-outs by reintegrating the Ura3 fragment into the target gene site to interrupt the gene transcription. The developed method allows to adapt sophisticate gene manipulation methods of model fungal species to non-model species.

中文翻译：

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体外合成的Cas9核糖核蛋白产生的红毛癣菌营养缺陷型突变。

背景技术毛癣毛癣菌是专性的人寄生真菌，负责人中约80-90％的皮肤癣菌病。该病原体的分子遗传操作具有挑战性，并且可用的工具和方案仅是基本的。我们采用完善的真菌模型生物的分子遗传学方法，以敲除红毛锥虫中的基因。为了适应，必须进行重要的修改。随着体外合成Cas9-sgRNA核糖核蛋白复合物的实施，有可能适应分子遗传学方法，以敲除红毛锥虫中的基因。结果基因敲除方法基于将选择标记整合到靶位点中，以中断基因翻译。靶基因由体外合成的Cas9-sgRNA核糖核蛋白复合物的同源序列预先分配。为了开发该方法，我们首先分离并鉴定了具有大量微分生孢子的红球菌菌株。接下来，我们开发了一种转化方案，即通过PEG方法将Cas9-sgRNA核糖核蛋白递送到真菌原生质体中。我们敲除了URA3基因，并产生了预期的尿嘧啶营养缺陷型菌株。通过重新整合URA3片段并在缺乏尿嘧啶的培养基中进行选择，这些菌株可用于特异性基因敲除。示例性地，我们敲除TRP3基因并得到了预测的表型色氨酸营养缺陷型菌株。该突变已通过测序验证。结论我们开发了一种方法，基于体外合成的Cas9-sgRNA核糖核蛋白复合物，用于风疹红菌中的靶标特异性基因敲除。我们敲除了Ura3基因并产生了尿嘧啶营养缺陷型菌株。通过将Ura3片段重新整合到目标基因位点以中断基因转录，将这些菌株用于目标特异性基因敲除。所开发的方法允许将模型真菌物种的复杂基因操纵方法适应于非模型物种。