ZFN protein-mediated genome editing of wheat microspores

Recent advances in genome engineering technologies based on designed endonucleases allow specific and predictable alterations in plant genomes to generate value added traits in the crops of choice. The EXZACTTM Precision technology, based on Zinc Finger Nucleases (ZFN), has been successfully used in the recent past for introduction of precise mutations and transgenes at specific genomic loci to generate novel and desired phenotypes in several crop species. Current methods for delivering ZFNs into plant cells are based on traditional genetic transformation methods that result in stable integration of the nuclease in the genome. Here we describe for the first time an alternative ZFN delivery method where plant cells are smartly transfected with ZFN protein that eliminates the need for stable nuclease genomic integration and allow to generate edited, but not genetically modified plants. For this, we complexed ZFN proteins purified from bacterial culture with cell penetrating peptides (CPP) and directly transfected the complex into either wheat microspores or embryos regenerated in vitro from isolated microspores. The IPK1 gene that encodes the inositol-1,3,4,5,6-pentakisphosphate 2-kinase was chosen as a target for disruption. This enzyme catalyzes the final steps of phytic acid synthesis that is an anti-nutritional component of feed grain. Next generation sequencing (NGS) analysis showed targeted edits at the IPK1 locus that were detected in the transfected samples. This is the first description of editing of microspores genome in plants by delivering designed endonuclease (DE) as a protein. The method has the promise of broad application in all crops amenable to microspore culture and to accelerate trait product development process.