Figure 3.

Overview of the Triple Cross tool for precise genetic manipulations. The triple cross tool relies on two counter-selectable markers (CS1 and CS2) in combination with one antibiotic marker (AB). CS1 and AB1 are located between two homology arms (5′ and 3′) together with a shorter, third homology arm (X). Using selection for AB and against CS1, a direct double crossover event at homology arms 3′ and 5′ is forced in a first step, this is facilitated by having homology arms of different length. In an optional second step, the marker can be recycled using shorter third homology arm X and selection against CS2. Shown is deletion of a target gene, but the same technology can also be used to deliver genes by placing the respective sequence between homology arms X and 3′. Depending how the homology arms are placed, the gene can either be inserted at any given position in the genome or an existing sequence be replaced by a new one.