Targeting DNA to a Previously Integrated Transgenic Locus Using Zinc Finger Nucleases

Targeting exogenously supplied DNA to a predetermined location within a plant genome provides a powerful tool for basic studies of plant gene function and opens up some intriguing possibilities for crop improvement. The induction of double-strand DNA breaks at specific genomic loci via the use of designed zinc finger nucleases (ZFNs) allows for targeted transgene integration. Preintegrating a reporter construct containing a nonfunctional herbicide resistance gene flanked by ZFN binding sites results in a locus capable of being targeted. Retransformation with a corresponding ZFN-expressing cassette and a donor DNA with sequences homologous to the integrated construct and capable of functionalizing the herbicide resistance gene following site-specific integration results in targeted DNA addition. Targeted DNA integration can be confirmed in herbicide-resistant plant cells using PCR analysis.