A recombineering-based platform for high-throughput genomic editing in Escherichia coli

Functional analysis of bacterial genes or genomic fragments in vivo 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 E. coli strains as well as wild-type strains such as E. coli MG1655, with particularly marked advantages demonstrated in E. coli 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 E. coli 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.