Enhancing the HoSeI method for one-step large-scale genome integration in Escherichia coli using multi-cutter plasmids

Precise chromosomal integration of large DNA fragments remains a significant challenge in metabolic engineering of Escherichia coli, largely due to the unpredictable and often low cleavage efficiency of individual single-guide RNAs (sgRNAs). To overcome this issue, we have developed a modular and versatile "multi-cutters" platform comprising plasmids designed to express up to four distinct sgRNAs simultaneously from a single vector. Building upon the HoSeI (Homologous Sequence Integration) method, this platform synergizes CRISPR-Cas9-mediated cleavage with λ-Red-mediated repair to enable one-step, scarless editing. Our construction strategy uses a modular assembly approach with specific linkers and restriction sites to enable the rapid and flexible generation of double, triple and quadruple cutters. We demonstrate the utility of this platform by successfully 'rewriting' genes—the one-step replacement of approximately 1 kbp chromosomal regions—by replacing the high-expression ompC and ompF loci with fluorescent reporter genes (mCherry and gfp). The resulting dual-reporter strain, RHA00802, functioned as an effective osmotic biosensor, exhibiting concentration-dependent fluorescence responses to sucrose levels (0–10%) in accordance with the regulation of the EnvZ–OmpR two-component system. This multi-cutter platform and gene rewriting strategy provides a powerful, generalized toolkit for complex genomic design and the rapid construction of functional microbial devices. (198 words)