Both authors should be considered co-first authors.
High-frequency homologous recombination in plants mediated by zinc-finger nucleases
Article first published online: 18 OCT 2005
The Plant Journal
Volume 44, Issue 4, pages 693–705, November 2005
How to Cite
Wright, D. A., Townsend, J. A., Winfrey, R. J., Irwin, P. A., Rajagopal, J., Lonosky, P. M., Hall, B. D., Jondle, M. D. and Voytas, D. F. (2005), High-frequency homologous recombination in plants mediated by zinc-finger nucleases. The Plant Journal, 44: 693–705. doi: 10.1111/j.1365-313X.2005.02551.x
- Issue published online: 18 OCT 2005
- Article first published online: 18 OCT 2005
- Received 28 June 2005; revised 3 August 2005; accepted 8 August 2005.
- gene targeting;
- genome engineering;
- double-strand breaks;
- non-homologous end joining
Homologous recombination offers great promise for plant genome engineering. This promise has not been realized, however, because when DNA enters plant cells homologous recombination occurs infrequently and random integration predominates. Using a tobacco test system, we demonstrate that chromosome breaks created by zinc-finger nucleases greatly enhance the frequency of localized recombination. Homologous recombination was measured by restoring function to a defective GUS:NPTII reporter gene integrated at various chromosomal sites in 10 different transgenic tobacco lines. The reporter gene carried a recognition site for a zinc-finger nuclease, and protoplasts from each tobacco line were electroporated with both DNA encoding the nuclease and donor DNA to effect repair of the reporter. Homologous recombination occurred in more than 10% of the transformed protoplasts regardless of the reporter's chromosomal position. Approximately 20% of the GUS:NPTII reporter genes were repaired solely by homologous recombination, whereas the remainder had associated DNA insertions or deletions consistent with repair by both homologous recombination and non-homologous end joining. The DNA-binding domain encoded by zinc-finger nucleases can be engineered to recognize a variety of chromosomal target sequences. This flexibility, coupled with the enhancement in homologous recombination conferred by double-strand breaks, suggests that plant genome engineering through homologous recombination can now be reliably accomplished using zinc-finger nucleases.