Cutting-Edge Methods and Tools for Plant Genome Editing

The development of DNA modifying enzymes with programmable DNA-binding specificities, derived from transcription activator-like effectors (TALEs) and the CRISPR/Cas9 system have opened new opportunities for more effective engineering of plant genomes. Using TALENs and CRISPR/Cas9 nucleases, gene knockouts can now be efficiently created in any plant species amenable to transformation. However, to fully exploit the potential of the gene editing technologies for synthetic biology, more advanced tools are needed to induce more complex modifications, such as gene replacement and multiplex gene editing, and to control transcriptional regulation. Moreover, parallel use of different gene editing techniques might be necessary to build and program genetic circuits and to make it possible to engineer whole genetic pathways. Here we introduce a comprehensive suite of methods and reagents that utilize both TALE(N)s and CRISPR/Cas9 for genome engineering in both monocot and dicot plant systems. The reagents are organized into an easy-to-use modular cloning system that offers the flexibility of combining different functional modules for different applications within a single construct. We have used this system to efficiently induce targeted gene insertions, to simultaneously edit multiple genes, and to create large deletions in a variety of plant species including tomato, wheat, barley, and cassava. Examples of successful genome engineering efforts will be presented.