Zhang Chi, Meng Xiuhua, Wei Xiaolei, Lu Ling
Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Microbiology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China.
Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Microbiology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China.
Fungal Genet Biol. 2016 Jan;86:47-57. doi: 10.1016/j.fgb.2015.12.007. Epub 2015 Dec 14.
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.
丝状真菌具有占主导地位的非同源末端连接（NHEJ）DNA修复途径，这导致大多数转化后代具有随机的异源插入诱变。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以探索发病机制。此外，没有任何选择营养标记且具有野生型ku80背景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，我们建立了一种高效的CRISPR诱变系统，通过非常短（约35个碱基对）的同源臂，以大约95 - 100%的准确率，在一个称为微同源介导的末端连接（MMEJ）的过程中，实现有或无标记插入的精确、高效的框内整合。基于该系统，我们成功地在预测位点高效、精确地整合了外源GFP标签且无标记插入，并在有或无选择标记插入的情况下，在多个预测位点编辑了分生孢子黑色素基因pksP和钙调神经磷酸酶基因cnaA的催化亚基cnaA。此外，我们发现MMEJ介导的CRISPR - Cas9诱变独立于ku80途径，这表明该系统可作为临床曲霉菌分离株中一种强大且通用的基因组编辑工具发挥作用。
