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.

中文翻译：

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使用限制-修饰系统编辑微生物基因组的新工具

基因组的无痕遗传操作是生物学研究的重要工具。限制性修饰（RM）系统是细菌中的防御系统，可根据其区分外源DNA与自身DNA的能力来防御入侵的基因组。在这里，我们设计了一种RM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REase的细菌，开发了一种利用可诱导DNA甲基转移酶基因bceSIIM（RMGE- bceSIIM）作为反选择盒的RMGE技术，以编辑大肠杆菌的基因组。对于没有IV型REase的细菌，已建立了基于限制性内切核酸酶（RMGE- mcrA）的RMGE技术。枯草芽孢杆菌。这些技术已成功用于基因缺失和替换，具有近100％的反选择效率，与传统方法相比，它们具有更高的稳定性。此外，使用RMGEbceSIIM在rpsL基因中引入了A128C的单碱基突变，从而在大肠杆菌中实现了无限制位点的精确点突变。另外，应用RMGE- mcrA技术以100％的反选择效率删除了酿酒酵母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 提示该技术对微生物基因组操作的潜在普遍用途。