Targeted mutagenesis in the progeny of maize transgenic plants
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We have demonstrated that targeted mutagenesis can be accomplished in maize plants by excision, activation, and subsequent elimination of an endonuclease in the progeny of genetic crosses. The yeast FLP/FRT site-specific recombination system was used to excise and transiently activate the previously integrated yeast I-SceI homing endonuclease in maize zygotes and/or developing embryos. An artificial I-SceI recognition sequence integrated into genomic DNA was analyzed for mutations to indicate the I-SceI endonuclease activity. Targeted mutagenesis of the I-SceI site occurred in about 1% of analyzed F1 plants. Short deletions centered on the I-SceI-produced double-strand break were the predominant genetic lesions observed in the F1 plants. The I-SceI expression cassette was not detected in the mutant F1 plants and their progeny. However, the original mutations were faithfully transmitted to the next generation indicating that the mutations occurred early during the F1 plant development. The procedure offers simultaneous production of double-strand breaks and delivery of DNA template combined with a large number of progeny plants for future gene targeting experiments.
KeywordsDouble-strand break I-SceI Mutation Transgenic plant Maize Zea mays L
The authors thank Shifu Zhen and Susan Nilles for technical assistance in producing transgenic plants. We acknowledge Carl Simmons’ contribution to this work by designing the maize codon-optimized I-SceI. We thank Cellectis, S.A. for providing the I-Sce endonuclease and for helpful research discussions.
Novel materials described in this publication may be available for non-commercial research purposes upon acceptance and signing of a material transfer agreement. In some cases such materials may contain or be derived from materials obtained from a third party. In such cases, distribution of material will be subject to the requisite permission from any third-party owners, licensors or controllers of all or parts of the material. Plant germplasm and transgenic material will not be made available except at the discretion of the owner and then only in accordance with all applicable governmental regulations. Obtaining any permissions will be the sole responsibility of the requestor.
- Arnould S, Perez C, Cabaniols JP, Smith J, Gouble A, Grizot S, Epinat JC, Duclert A, Duchateau P, Paques F (2007) Engineered I-CreI derivatives cleaving sequences from the human XPC gene can induce highly efficient gene correction in mammalian cells. J Mol Biol 371:49–65. doi: 10.1016/j.jmb.2007.04.079 PubMedCrossRefGoogle Scholar
- Cheng P-C, Pareddy DR (1994) Morphology and development of the tassel and ear. In: Freeling M, Walbot V (eds) The maize handbook. Springer-Verlag, New York, Berlin, Heidelberg, London, Paris, pp 37–47Google Scholar
- Djukanovic V, Orczyk W, Gao H, Sun X, Garrett N, Zhen S, Gordon-Kamm W, Barton J, Lyznik LA (2006) Gene conversion in transgenic maize plants expressing FLP/FRT and Cre/loxP site-specific recombination systems. Plant Biotechnol J 4:345–357. doi: 10.1111/j.1467-7652.2006.00186.x PubMedCrossRefGoogle Scholar
- Doyon Y, McCammon JM, Miller JC, Faraji F, Ngo C, Katibah GE, Amora R, Hocking TD, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Amacher SL (2008) Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat Biotechnol 26:702–708. doi: 10.1038/nbt1409 PubMedCrossRefGoogle Scholar
- Gao H, Yang M, Falco SC, Lyznik A (2008) Locus-specific mutations induced by homing endonucleases in maize. FAO/IAEA International Symposium on induced mutations in plants. Vienna, Austria, pp 86Google Scholar
- Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Anthony RM, Eichtinger M, Jiang T, Foley JE, Winfrey RJ, Townsend JA, Unger-Wallace E, Sander JD, Muller-Lerch F, Fu F, Pearlberg J, Gobel C, Dassie JP, Pruett-Miller SM, Porteus MH, Sgroi DC, Iafrate AJ, Dobbs D, McCray PB Jr, Cathomen T, Voytas DF, Joung JK (2008) Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell 31:294–301. doi: 10.1016/j.molcel.2008.06.016 PubMedCrossRefGoogle Scholar
- Redondo P, Prieto J, Munoz IG, Alibes A, Stricher F, Serrano L, Cabaniols JP, Daboussi F, Arnould S, Perez C, Duchateau P, Paques F, Blanco FJ, Montoya G (2008) Molecular basis of Xeroderma pigmentosum group C DNA recognition by engineered meganucleases. Nature 456:107–111. doi: 10.1038/nature07343 PubMedCrossRefGoogle Scholar