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.
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