Development of a marker-free mutagenesis system using CRISPR-Cas9 in the pathogenic mould Aspergillus fumigatus.
Development of a marker-free mutagenesis system using CRISPR-Cas9 in the pathogenic mould Aspergillus fumigatus.
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DOI：
10.1016/j.fgb.2020.103479
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发表时间：
2020-12
期刊：
影响因子：
--
通讯作者：
Bromley MJ
中科院分区：
文献类型：
--
作者：
van Rhijn N;Furukawa T;Zhao C;McCann BL;Bignell E;Bromley MJ
体外组装的CRISPR/Cas9-gRNA复合物可以介导无选择转化。单个gRNA可用于有效地靶向诱变。靶向点突变可以利用单链DNA寡核苷酸作为修复模板来实现。烟曲霉是一种致病性真菌病原体，每年导致超过30万例危及生命的感染。我们对发病机制和导致疾病进展的因素的理解是有限的。开发快速和通用的A.烟熏是必不可少的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，如A.烟熏。然而，成功的基因组编辑依赖于耗时的多步克隆程序，并与选择标记的使用配对，这可能导致宿主的代谢负担和/或整合位点处的非预期转录修饰。我们使用体外CRISPR-Cas9组装方法来进行无选择基因组编辑，包括蛋白质的表位标记和定点诱变。在该转化过程中使用的修复模板使用50 bp微同源臂，并且可以用单个PCR反应或通过购买合成的单链寡核苷酸来产生，从而减少复杂构建体合成所需的时间。
In vitro assembled CRISPR/Cas9-gRNA complexes can mediate selection free transformation. Single gRNAs can be used to target mutagenesis effectively. Targeted point mutations can be achieved utilising single strand DNA oligonucleotides as repair template. 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.
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影响因子：
2.9
作者：
Bertuzzi M;van Rhijn N;Krappmann S;Bowyer P;Bromley MJ;Bignell EM
通讯作者：
Bignell EM
影响因子：
4.6
作者：
Foster AJ;Martin-Urdiroz M;Yan X;Wright HS;Soanes DM;Talbot NJ
通讯作者：
Talbot NJ
影响因子：
3
作者：
Nodvig, Christina S.;Hoof, Jakob B.;Mortensen, Uffe H.
通讯作者：
Mortensen, Uffe H.
影响因子：
4.1
作者：
Liang, Xiquan;Potter, Jason;Chesnut, Jonathan D.
通讯作者：
Chesnut, Jonathan D.
影响因子：
7
作者：
Kim S;Kim D;Cho SW;Kim J;Kim JS
通讯作者：
Kim JS