Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus fumigatus
Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus fumigatus
复制标题
通过微同源介导的烟曲霉末端连接实现高效 CRISPR 诱变
DOI：
10.1016/j.fgb.2015.12.007
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发表时间：
2016-01-01
影响因子：
3
通讯作者：
Lu, Ling
中科院分区：
文献类型：
--
作者：
Zhang, Chi;Meng, Xiuhua;Lu, Ling
丝状真菌具有显性的非同源末端连接(NHEJ)DNA修复途径，导致大多数转化后代具有随机的异源插入突变。因此，缺乏通用的基因组编辑工具阻碍了我们进行精确的基因组编辑来探索发病机制。此外，具有野生型ku80背景而没有任何选择营养标记的临床分离株尤其存在同源整合效率低的问题。在本研究中，我们建立了一个高效的CRISPR突变系统，在有或没有标记插入的情况下，通过非常短(约35bp)的同源臂，在被称为微同源介导的末端连接(MMEJ)的过程中，以大约95%-100%的准确率进行精确和高效的框内整合。基于该系统，我们成功地实现了外源GFP标签在无标记插入的预测位点上的高效精确整合，并在有或没有选择标记插入的多个预测位点编辑了分生黑色素基因pksP和钙调神经磷酸酶基因的催化亚基。此外，我们发现MMEJ介导的CRISPR-Cas9突变不依赖于ku80途径，这表明该系统可以作为一种强大而通用的基因组编辑工具用于临床曲霉分离株。(C)2015 Elsevier Inc.保留所有权利。
Filamentous fungi have a dominant nonhomologous-end joining (NHEJ) DNA repair pathway, which results in the majority of transformed progenies having random heterologous insertion mutagenesis. Thus, lack of a versatile genome-editing tool prevents us from carrying out precise genome editing to explore the mechanism of pathogenesis. Moreover, clinical isolates that have a wild-type ku80 background without any selection nutrition marker especially suffer from low homologous integration efficiency. In this study, we have established a highly efficient CRISPR mutagenesis system to carry out precise and efficient in-frame integration with or without marker insertion with approximately 95-100% accuracy via very short (approximately 35-bp) homology arms in a process referred to as microhomology-mediated end joining (MMEJ). Based on this system, we have successfully achieved an efficient and precise integration of an exogenous GFP tag at the predicted site without marker insertion and edited a conidial melanin gene pksP and a catalytic subunit of calcineurin gene cnaA at multiple predicted sites with or without selection marker insertion. Moreover, we found that MMEJ-mediated CRISPR-Cas9 mutagenesis is independent of the ku80 pathway, indicating that this system can function as a powerful and versatile genome-editing tool in clinical Aspergillus isolates. (C) 2015 Elsevier Inc. All rights reserved.