Aspergillus fumigatus is a saprophytic fungal pathogen that is the cause of more than 300,000 life-threatening infections annually. Our understanding of pathogenesis and factors contributing to disease progression are limited. Development of rapid and versatile gene editing methodologies for A. fumigatus is essential. CRISPR-Cas9 mediated transformation has been widely used as a novel genome editing tool and has been used for a variety of editing techniques, such as protein tagging, gene deletions and site-directed mutagenesis in A. fumigatus. However, successful genome editing relies on time consuming, multi-step cloning procedures paired with the use of selection markers, which can result in a metabolic burden for the host and/or unintended transcriptional modifications at the site of integration. We have used an in vitro CRISPR-Cas9 assembly methodology to perform selection-free genome editing, including epitope tagging of proteins and site-directed mutagenesis. The repair template used during this transformation use 50 bp micro-homology arms and can be generated with a single PCR reaction or by purchasing synthesised single stranded oligonucleotides, decreasing the time required for complex construct synthesis.

烟曲霉是一种腐生真菌病原体，每年导致超过 300,000 例危及生命的感染。我们对发病机制和导致疾病进展的因素的了解是有限的。为A. fumigatus开发快速和通用的基因编辑方法是必不可少的。CRISPR-Cas9 介导的转化已被广泛用作新型基因组编辑工具，并已用于多种编辑技术，例如烟曲霉中的蛋白质标记、基因删除和定点诱变. 然而，成功的基因组编辑依赖于耗时的多步骤克隆程序以及选择标记的使用，这可能导致宿主的代谢负担和/或整合位点的意外转录修饰。我们使用体外CRISPR-Cas9 组装方法进行无选择基因组编辑，包括蛋白质的表位标记和定点突变。在此转化过程中使用的修复模板使用 50 bp 微同源臂，可以通过单个 PCR 反应或通过购买合成的单链寡核苷酸生成，从而减少复杂结构合成所需的时间。