The comparison of seamless genome editing with traditional HR-based marker selection. (a) Traditional HR. (b) Seamless genome editing. Homology arms (dark grey and light grey boxes) bearing the desired mutation (red bar) are used to flank an excisable selection marker cassette. This is achieved by using the tandem loxP sites as in (a) and a PiggyBac transposon as in (b). Successful HR will insert the selection marker cassette into the genome (middle panels). Removing the loxP cassette with Cre recombinase will leave one loxP site at the locus of interest (blue triangle) in (a). The remobilization of the PiggyBac transposon will only leave a “TA” dinucleotide in (b), which initially can be found in the locus of interest, or can be tolerated without any undesired changes to the protein sequence.