A Simple Way to Detect in Planta ZFN Activity and Chimerism in Gene Targeting

In planta gene targeting (GT), via double strand breaks (DSB) using site specific nucleases has become increasingly attractive for trait product development due to its independence of transformation efficiency. However, DSB-induced GT through breeding crosses often results in chimeric F1 tissues. Reporter genes, such as GUS or GFP, combined with histochemical staining have been used frequently to visualize the mutations as a proof of concept. For commercial trait product development, these reporter gene systems may not be suitable. Here we report the use of a simple quantitative PCR based method to detect the activity of zinc finger nucleases (ZFN). Each ZFN recognizes only one DNA binding site and create one DSB. During the process of DSB repair, the plant cells make small deletions and/or insertions on specific ZFN recognition sequences. As a measurement for relative ZFN activity, qPCR based disruption assays were used to detect the imperfect repairs. Spatial/temporal differences in somatic and meristem tissue were also detected using this methodology.