Liang Zeyu, Huang Chaoyong, Li Yitian, Yang Chao, Wang Ning, Ma Xiaoyan, Huo Yi-Xin
Center for Future Foods, Muyuan Laboratory, Zhengzhou, Henan Province, China.
Key Laboratory of Molecular Medicine and Biotherapy, Aerospace Center Hospital, School of Life Science, Beijing Institute of Technology, Beijing, China.
Appl Environ Microbiol. 2025 Jul 23;91(7):e0019325. doi: 10.1128/aem.00193-25. Epub 2025 Jun 12.
Functional analysis of bacterial genes or genomic fragments primarily relies on the analysis of knockout strains. Although various methods have successfully generated bacterial knockout mutants, the parallel operation of multiple sites, especially in biofoundries, remains challenging. New technological refinements of existing methods are necessary for high-throughput genomic editing in bacteria. In this study, to modify numerous sites in parallel, we optimized the linear donor DNA by adding modification at the different positions and achieved high-efficiency recombination with chemical transformation. Then, by combining with the CRISPR system, we established a guide sequence-independent and donor DNA-mediated genomic editing (GIDGE) method, enabling efficient and scarless engineering of common strains as well as wild-type strains such as MG1655, with particularly marked advantages demonstrated in Nissle 1917. This method allows for high-throughput genomic engineering in a 96-well format and is useful for sequence deletion with a wide range of lengths, sequence insertion, sequence replacement, and point mutation. As a proof-of-concept study, we constructed 96 single-gene knockout mutants and a genomic large-fragment deletion library in K-12 MG1655 using the GIDGE method. This high-throughput and easy-to-use method has great potential for automation and can be adapted for use in biofoundries.
With the increasing demand in the microbiology field and the expansion of its application scope, the urgency for genome editing techniques that are not only efficient and versatile but also capable of high-throughput processing and even automation has become increasingly critical. In this study, we enhanced the efficiency of recombination engineering by incorporating modifications and integrated it with the CRISPR system to develop an advanced gene editing method. This method allows for various gene editing events such as insertion, replacement, and long fragment knockout without the need for plasmid construction. It not only demonstrated high efficiency in common strains but also exhibited marked advantages in the probiotic strain Nissle 1917. This method is a versatile, efficient approach capable of high-throughput parallel gene editing. Using this method, we successfully constructed a large-scale strain library, significantly accelerating the process of microbial engineering.
细菌基因或基因组片段的功能分析主要依赖于对基因敲除菌株的分析。尽管各种方法已成功产生细菌基因敲除突变体，但多个位点的并行操作，尤其是在生物铸造厂中，仍然具有挑战性。现有方法的新技术改进对于细菌的高通量基因组编辑是必要的。在本研究中，为了并行修饰多个位点，我们通过在不同位置添加修饰来优化线性供体DNA，并通过化学转化实现了高效重组。然后，通过与CRISPR系统相结合，我们建立了一种不依赖引导序列且由供体DNA介导的基因组编辑（GIDGE）方法，能够对常见菌株以及野生型菌株（如MG1655）进行高效且无痕的工程改造，在Nissle 1917中表现出尤为显著的优势。该方法允许以96孔板形式进行高通量基因组工程，可用于各种长度的序列删除、序列插入、序列替换和点突变。作为概念验证研究，我们使用GIDGE方法在K - 12 MG1655中构建了96个单基因敲除突变体和一个基因组大片段缺失文库。这种高通量且易于使用的方法具有很大的自动化潜力，可适用于生物铸造厂。
随着微生物学领域需求的增加及其应用范围的扩大，对不仅高效通用而且能够进行高通量处理甚至自动化的基因组编辑技术的迫切需求变得越来越关键。在本研究中，我们通过引入修饰提高了重组工程的效率，并将其与CRISPR系统整合以开发一种先进的基因编辑方法。该方法允许进行各种基因编辑事件，如插入、替换和长片段敲除，而无需构建质粒。它不仅在常见菌株中显示出高效率，而且在益生菌菌株Nissle 1917中也表现出显著优势。该方法是一种通用、高效的方法，能够进行高通量并行基因编辑。使用该方法，我们成功构建了一个大规模菌株文库，显著加速了微生物工程的进程。
