Targeted transgene integration in plant cells using designed zinc finger nucleases

Journal Article / 2008

Cai, Charles Q.; Doyon, Yannick; Ainley, W. Michael; Miller, Jeffrey C.; DeKelver, Russell C.; Moehle, Erica A.; Rock, Jeremy M.; Lee, Ya-Li; Garrison, Robbi; Schulenberg, Lisa; Blue, Ryan; Worden, Andrew; Baker, Lisa; Faraji, Farhoud; Zhang, Lei; Holmes, Michael C.; Rebar, Edward J.; Collingwood, Trevor N.; Rubin-Wilson, Beth; Gregory, Philip D.; Urnov, Fyodor D.; Petolino, Joseph F.

Targeted transgene integration in plants remains a significant technical challenge for both basic and applied research. Here it is reported that designed zinc finger nucleases (ZFNs) can drive site-directed DNA integration into transgenic and native gene loci. A dimer of designed 4-finger ZFNs enabled intra-chromosomal reconstitution of a disabled gfp reporter gene and site-specific transgene integration into chromosomal reporter loci following co-transformation of tobacco cell cultures with a donor construct comprised of sequences necessary to complement a non-functional pat herbicide resistance gene. In addition, a yeast-based assay was used to identify ZFNs capable of cleaving a native endochitinase gene. Agrobacterium delivery of a Ti plasmid harboring both the ZFNs and a donor DNA construct comprising a pat herbicide resistance gene cassette flanked by short stretches of homology to the endochitinase locus yielded up to 10% targeted, homology-directed transgene integration precisely into the ZFN cleavage site. Given that ZFNs can be designed to recognize a wide range of target sequences, these data point toward a novel approach for targeted gene addition, replacement and trait stacking in plants.