Highly efficient single base editing in Aspergillus niger with CRISPR/Cas9 cytidine deaminase fusion
Highly efficient single base editing in Aspergillus niger with CRISPR/Cas9 cytidine deaminase fusion
复制标题
利用 CRISPR/Cas9 胞苷脱氨酶融合对黑曲霉进行高效单碱基编辑
DOI：
10.1016/j.micres.2019.03.007
复制
发表时间：
2019-06-01
影响因子：
6.7
通讯作者：
Pan, Li
中科院分区：
文献类型：
--
作者：
Huang, Lianggang;Dong, Hongzhi;Pan, Li
包括ZFN、TALEN和CRISPR/Cas9在内的经典基因组编辑工具依赖于DNA双链断裂进行基因组编辑。为了防止双链断裂（DSB）所带来的潜在危害，已经在多个物种中广泛开发了一系列将胞苷（C）转化为胸腺嘧啶（T）而没有DSB的单碱基编辑工具。在此，我们首次报道了通过融合胞苷脱氨酶和Cas9切口酶，在丝状真菌尼日尔曲霉中C以高频率转化为T。使用CRISPR/Cas9依赖性碱基编辑器并通过单碱基编辑诱导无义突变，我们在黑曲霉中以47.36%-100%的效率灭活了尿苷营养缺陷型基因pyrG和色素基因fwnA。同时，非表型基因prtT的单碱基编辑结果显示效率为60%。在A中，可编辑窗口达到8个碱基（在原型间隔区中从C2到C9）。尼日尔.总体上，我们成功地构建了一个A.尼日尔.该系统为研究A.尼日尔，并为丝状真菌的遗传修饰提供了新的工具。
Classic genome editing tools including ZFN, TALEN, and CRISPR/Cas9 rely on DNA double-strand breaks for genome editing. To prevent the potential hazard caused by double-strand breaks (DSBs), a series of single base editing tools that convert cytidine (C) to thymine (T) without DSBs have been developed extensively in multiple species. Herein, we report for the first time that C was converted to T with a high frequency in the filamentous fungi Aspergillus niger by fusing cytidine deaminase and Cas9 nickase. Using the CRISPR/Cas9-dependent base editor and inducing nonsense mutations via single base editing, we inactivated the uridine auxotroph gene pyrG and the pigment gene fwnA with an efficiency of 47.36%-100% in A.niger. At the same time, the single-base editing results of the non-phenotypic gene prtT showed an efficiency of 60%. The editable window reached 8 bases (from C2 to C9 in the protospacer) in A. niger. Overall, we successfully constructed a single base editing system in A. niger. This system provides a more convenient tool for investigating gene function in A. niger, and provides a new tool for genetic modification in filamentous fungi.