Tian Li, Yan Bingyu, Huo Dandan, Sun Wenhui, Cui Sufang, Li Xiaojing, Zhang Xiangmei, Dong Huijun
School of Pharmaceutical Sciences, Liaocheng University, No.1 Hunan Road, Dongchangfu District, Liaocheng, 252000, China.
Biotechnol Lett. 2023 Mar;45(3):401-410. doi: 10.1007/s10529-023-03347-1. Epub 2023 Jan 18.
To develop a modified CRISPR/Cas9 system with the β-glucuronidase (GusA) reporter and a dual sgRNA cassette for Nonomuraea gerenzanensis (N. gerenzanensis).
With the aid of a visual GusA reporter, the complicated and tedious process of cloning and gene identification could be abandoned entirely in the genetic editing of N. gerenzanensis. Moreover, introducing a dual sgRNA cassette into the CRISPR/Cas9 system significantly improved gene deletion efficiency compared to the single sgRNA element. Furthermore, the length of the homologous flanking sequences set to the lowest value of 500 bp in this system could still reach the relatively higher conjugation transfer frequency.
The enhanced CRISPR/Cas9 system could efficiently perform genetic manipulation on the rare actinomycete N. gerenzanensis.
开发一种带有β-葡萄糖醛酸酶（GusA）报告基因和双sgRNA盒的改良CRISPR/Cas9系统，用于热泉野野村菌（N. gerenzanensis）。
借助可视化的GusA报告基因，在热泉野野村菌的基因编辑中可以完全摒弃复杂繁琐的克隆和基因鉴定过程。此外，与单sgRNA元件相比，在CRISPR/Cas9系统中引入双sgRNA盒显著提高了基因缺失效率。此外，该系统中同源侧翼序列的长度设定为最低值500 bp时，仍可达到相对较高的接合转移频率。
增强型CRISPR/Cas9系统能够有效地对稀有放线菌热泉野野村菌进行基因操作。
