Fig. 2

Schematic overview of genome editing methods based on counter-selectable markers. Left: genome editing (gene knockout as an example) with two integration steps. Step 1, an exogenous artificial DNA (plasmid or fragment) with up- and downstream homologous sequences is integrated into the genome, replacing the target gene. The recombinant clone can be selected under condition A. Step 2, under the selection condition B, the clone obtained in step 1 deleted the selectable marker and repressor/toxin gene through a self-recombination with the DR (direct repeats). Right: Composition of the selectable elements, the selectable marker A, toxin gene/repressor, and up- and downstream homologous sequences of the target gene can be constructed as fragments or on a plasmid. Examples of selectable markers A include: cat (chloramphenicol), phleo (phleomycin), or spe (spectinomycin). Examples of toxic genes include: upp, pyrF, or mazF. Examples of repressor genes include: xylR, blaI, araR, or lacI. The repressor can inhibit the expression of the selectable marker B, which can be integrated into the genome or a plasmid