Selection-based recombineering is a flexible and proven technology to precisely modify bacterial genomes at single base resolution. It consists of two steps of homologous recombination followed by selection/counter-selection. However, the shortage of efficient counterselectable markers limits the throughput of this method. Additionally, the emergence of 'selection escapees' can affect recombinant pools generated through this method, and they must be manually removed at each step of selection-based recombineering. Here, we report a series of efforts to improve the throughput and robustness of selection-based recombineering and to achieve seamless and automatable genome engineering. Using the nucleoside kinase activity of herpes simplex virus thymidine kinase (hsvTK) on the non-natural nucleoside dP, a highly efficient, rapid, and liquid-based counter-selection system was established. By duplicating hsvtk gene, combined with careful control of the population size for the subsequent round, we effectively eliminated selection escapes, enabling seamless and multiple insertions/replacement of gene-size fragments in the chromosome. Four rounds of recombineering could thus be completed in 10 days, requiring only liquid handling and without any need for colony isolation or genotype confirmation. The simplicity and robustness of our method make it broadly accessible for multi-locus chromosomal modifications.
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