Fig. 1

Gene stacking approach based on the use of Cre-lox for gene integration and a pair of nucleases for marker deletion. a Gene stacking site consists of a loxP fused to promoter-less marker gene (NPT*) followed by the first nuclease target site, e.g., I-SceI site. The GS construct could also include the first set of gene(s)-of-interest (GOI-1); b The first donor vector, pDonor1, contains loxP flanked construct consisting of the second promoter-less marker gene (HPT*), the second nuclease target site, e.g., ZFN site, GOI-2, the first nuclease target site, and the promoter for activating NPT gene at the GS site; c Co-delivery of pDonor1 and cre gene generates site-specific integration (SSI) locus, which is selectable due to the activation of NPT gene, and contains two I-SceI target sites for marker excision; d Delivery of I-SceI gene into SSI cells leads to excision of NPT gene, seamlessly connecting GOI-1 and GOI-2; e The second donor vector, pDonor2, is similar to pDonor1 except the first marker gene, NPT, is included and nuclease target sites are exchanged. Targeting of pDonor2 into marker-free SSI-1 line will generate SSI-2 that is selectable due to HPT activation, and converted to marker-free SSI-2 by ZFN activity