counterselectable

Counterselectable refers to genetic elements or markers that impose a disadvantage under a specific selective condition, so that organisms carrying the element cannot grow, while variants that have lost it can thrive. In molecular genetics, counterselection is used to remove or replace sequences during processes such as allelic exchange, plasmid curing, or genome editing. The typical workflow involves integrating a counterselectable marker with a desired modification, selecting for organisms that retain the marker under permissive conditions, and then applying counterselective pressure so that only cells that have lost the marker (and thus incorporated the desired change) survive.

Common counterselectable systems include sacB, which encodes levansucrase; in many Gram-negative bacteria, expression of SacB in the presence of sucrose is lethal, allowing selection against those cells. Other examples include galK or upp-based systems in bacteria, where growth on media containing 2-deoxy-galactose or 5-fluorouracil selects for loss of the marker. In yeast, URA3 together with 5-FOA enables counterselection, because cells expressing URA3 convert 5-FOA into a toxic compound, so only cells lacking URA3 survive on 5-FOA.

Applications of counterselectable markers include facilitating precise genetic modifications, such as seamless allelic replacement, removal of selectable markers after editing, and genome editing workflows where one wants to enrich for cells that have undergone a specific genetic change. Limitations include background growth, variable efficiency across species or strains, and the potential for escape mutants. Counterselectable markers are often used alongside positive selection markers to improve overall editing efficiency.