Non-transgenic Plant Genome Editing Using Purified Sequence-Specific Nucleases

Journal Article / 2015

Luo, Song; Li, Jin; Stoddard, Thomas J.; Baltes, Nicholas J.; Demorest, Zachary L.; Clasen, Benjamin M.; Coffman, Andrew; Retterath, Adam; Mathis, Luc; Voytas, Daniel F.; Zhang, Feng

Sequence-specific nucleases, including zinc-finger nucleases, meganucleases, TAL effector nucleases (TALENs), and CRISPR/ Cas systems, have been used to introduce targeted mutations in a wide range of plant species (Voytas, 2013; Baltes and Voytas, 2015). However, delivery of these nucleases using traditional transformation methods (e.g., particle bombardment, Agrobacterium or protoplast transformation) may result in undesired genetic alterations due to random insertion of nucleaseencodingDNAinto thehost genome. Randomintegration ofDNA is a particular concern for trait improvement and gene function studies, because it may lead to unintended gene inactivation or it may alter expression of host genes. Furthermore, for crop varieties created using biotechnology, the presence of foreign DNA is a trigger for regulation by many governmental agencies (Voytas and Gao, 2014). Therefore, methods to modify plant genomes that do not require DNA delivery would have value in both commercial and academic settings. Here we demonstrate nontransgenic plant genome engineering by introducing sequencespecific nucleases as purified protein. This approach enabled targetedmutagenesis ofendogenoussequenceswithinplant cells, while avoiding integration of foreign DNA into the genome.