ZFN-Mediated TLP Targeting in Maize Inbred Line B104

A simple approach for trait stacking had been demonstrated in a ‘Hi-II’ maize genotype via zinc finger nucleases (eZFNs) mediated targeted transgene integration (Ainley et al., 2013).  ‘Hi-II’ is a hybrid germplasm with relatively high transformation efficiency.  However, it is not optimal for crop improvement due to the segregation of multiple genes by virtue of its hybrid nature along with its inferior agronomic performance.  The same gene targeting strategy was applied to a maize inbred line, B104.  Target lines were constructed by first integrating two genes of interest (GOI), a selectable marker (aad1) and an ‘ExZACT™ trait anding pad’ (TLP) containing ZFN recognition sites and sequences homologous to incoming DNA.  Microparticle bombardment was used to deliver a donor construct, with the pat selectable marker gene flanked by sequences homologous to the integrated TLP, along with an eZFN-expressing constructs into immature embryos of the B104 target lines.  Following eZFN cleavage at the TLP and targeted double stranded break formation, the donor DNA was integrated through homology-directed repair.  Three ‘perfect’ four gene molecular stacks were created, demonstrating a 0.9% retargeting frequency.  This study exemplifies ZFN-mediated TLP targeting in a inbred maize germplasm.