Zinc finger nuclease and homing endonuclease-mediated assembly of multigene plant transformation vectors.

Binary vectors are an indispensable component of modern Agrobacterium tumefaciens-mediated plant genetic transformation systems. A remarkable variety of binary plasmids have been developed to support the cloning and transfer of foreign genes into plant cells. The majority of these systems, however, are limited to the cloning and transfer of just a single gene of interest. Thus, plant biologists and biotechnologists face a major obstacle when planning the introduction of multigene traits into transgenic plants. Here, we describe the assembly of multitransgene binary vectors by using a combination of engineered zinc finger nucleases (ZFNs) and homing endonucleases. Our system is composed of a modified binary vector that has been engineered to carry an array of unique recognition sites for ZFNs and homing endonucleases and a family of modular satellite vectors. By combining the use of designed ZFNs and commercial restriction enzymes, multiple plant expression cassettes were sequentially cloned into the acceptor binary vector. Using this system, we produced binary vectors that carried up to nine genes. Arabidopsis (Arabidopsis thaliana) protoplasts and plants were transiently and stably transformed, respectively, by several multigene constructs, and the expression of the transformed genes was monitored across several generations. Because ZFNs can potentially be engineered to digest a wide variety of target sequences, our system allows overcoming the problem of the very limited number of commercial homing endonucleases. Thus, users of our system can enjoy a rich resource of plasmids that can be easily adapted to their various needs, and since our cloning system is based on ZFN and homing endonucleases, it may be possible to reconstruct other types of binary vectors and adapt our vectors for cloning on multigene vector systems in various binary plasmids.