Bai Hua, Deng Aihua, Liu Shuwen, Cui Di, Qiu Qidi, Wang Laiyou, Yang Zhao, Wu Jie, Shang Xiuling, Zhang Yun, Wen Tingyi
CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences , Beijing 100101, China.
University of Chinese Academy of Sciences , Beijing 100049, China.
ACS Synth Biol. 2018 Jan 19;7(1):98-106. doi: 10.1021/acssynbio.7b00254. Epub 2017 Oct 12.
Scarless genetic manipulation of genomes is an essential tool for biological research. The restriction-modification (R-M) system is a defense system in bacteria that protects against invading genomes on the basis of its ability to distinguish foreign DNA from self DNA. Here, we designed an R-M system-mediated genome editing (RMGE) technique for scarless genetic manipulation in different microorganisms. For bacteria with Type IV REase, an RMGE technique using the inducible DNA methyltransferase gene, bceSIIM (RMGE-bceSIIM), as the counter-selection cassette was developed to edit the genome of Escherichia coli. For bacteria without Type IV REase, an RMGE technique based on a restriction endonuclease (RMGE-mcrA) was established in Bacillus subtilis. These techniques were successfully used for gene deletion and replacement with nearly 100% counter-selection efficiencies, which were higher and more stable compared to conventional methods. Furthermore, precise point mutation without limiting sites was achieved in E. coli using RMGE-bceSIIM to introduce a single base mutation of A128C into the rpsL gene. In addition, the RMGE-mcrA technique was applied to delete the CAN1 gene in Saccharomyces cerevisiae DAY414 with 100% counter-selection efficiency. The effectiveness of the RMGE technique in E. coli, B. subtilis, and S. cerevisiae suggests the potential universal usefulness of this technique for microbial genome manipulation.
基因组的无痕遗传操作是生物学研究的一项重要工具。限制修饰（R-M）系统是细菌中的一种防御系统，它基于区分外源DNA和自身DNA的能力来抵御入侵的基因组。在此，我们设计了一种R-M系统介导的基因组编辑（RMGE）技术，用于在不同微生物中进行无痕遗传操作。对于具有IV型限制酶的细菌，开发了一种使用可诱导DNA甲基转移酶基因bceSIIM（RMGE-bceSIIM）作为反选择盒的RMGE技术，用于编辑大肠杆菌的基因组。对于没有IV型限制酶的细菌，在枯草芽孢杆菌中建立了一种基于限制性内切酶的RMGE技术（RMGE-mcrA）。这些技术成功用于基因缺失和替换，反选择效率近100%，与传统方法相比更高且更稳定。此外，使用RMGE-bceSIIM在大肠杆菌中实现了无限制位点的精确点突变，将rpsL基因中的A128C单碱基突变引入其中。此外，RMGE-mcrA技术以100%的反选择效率应用于删除酿酒酵母DAY414中的CAN1基因。RMGE技术在大肠杆菌、枯草芽孢杆菌和酿酒酵母中的有效性表明了该技术在微生物基因组操作中潜在的普遍实用性。
