Fig. 1.

Fig. 1. Genome engineering methods in bacteria. (A) Genome editing using counter-selectable markers (CSM). The first single crossover results during the integration of suicide plasmids. The second crossover can be selected via counterselection (e.g., sucrose in the media is toxic to cells when the sacB gene is present in the genome), which results in the excision of the plasmid backbone. Editing and non-editing events have an equal likelihood of occurrence (i.e., a fifty-fifty chance). (B) Scarless gene deletion. Three different homologous DNA fragments were rearranged, and an antibiotic resistance marker (Ab^R) flanking I-SceI cleavage sites was inserted in the middle of three PCR-fused DNA fragments. The chromosomal DNA sequences to be excised were deleted via HR without any traces or scar sequences after I-SceI cleavage. The dotted lines indicate the homologous recombination between chromosomal DNA and PCR products. Red arrows indicate an I-SceI cleavage. (C) CRISPR-mediated genome editing. Phage recombinases facilitate the integration of donor templates (ss oligos or dsDNA) into the chromosomal DNA. Unedited DNA targets are recognized and cleaved by the guide RNA/Cas nuclease complex. Unedited cells die, as most bacteria lack the DSB repair system. Finally, edited cells are obtained via CRISPR/Cas negative selection.

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