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FIGS. 15A and 15B are schematics depicting exogenous marker-free, sequential transgene stacking at an endogenous AHAS locus in the wheat genome of Triticum aestivum using ZFN-mediated, HDR-directed DNA repair. FIGS. 14A and 14B are schematics depicting exogenous marker-view free porn Video, sequential transgene stacking at an endogenous AHAS locus in the wheat genome of Triticum aestivum using ZFN-mediated, NHEJ-directed DNA repair. In an embodiment, the AHAS gene is located on an A, B, or D genome of a polyploidy genome. In a further embodiment, the modification disrupts expression of the endogenous gene. In further embodiments, the integration of the transgene further comprises introduction of one or more indels that disrupt expression of the one or more endogenous loci and produce the selectable phenotype. In other embodiments, the whole plant comprising the one or more exogenous sequences integrated within the one or more endogenous loci of the plant genome is further modified to incorporate an additional exogenous sequence within the endogenous loci of the plant gen
nIn further embodiments, the one or more endogenous loci comprise an acetohydroxyacid synthase (AHAS) gene. In an embodiment, the endogenous gene is an acetohydroxyacid synthase (AHAS) gene. Other methods of measuring gene and/or encoded polypeptide activity can be used. The methods and compositions described herein are advantageous in providing targeted integration into a selected locus without the use of an exogenous transgenic marker to assess integration. Integration of the exogenous sequence(s) (e.g., into the AHAS locus) is facilitated by targeted double-strand cleavage of endogenous sequence, for example by cleavage of a sequence located in the 3′ untranslated region. Thus, the present disclosure provides for exogenous sequence integration, including transgene stacking, into an endogenous locus, which endogenous locus can serve as a marker for integration (e.g., the AHAS locus in which single mutations can impart herbicide tolerance). Integration of exogenous sequences can proceed through both homology-dependent and homology-independent mechanisms, and the selection of precisely targeted events is achieved through screening for a selectable marker (e.g., tolerance to a specific Group B herbicide, or ALS inhibitor herbicides such as imidazolinone or sulfonylurea) which is only functional in correctly targeted events. In a subsequent embodiment, the integration of the one or more exogenous sequences occurs by homologous recombination or non-homologous end join
n In a still further aspect, a plant cell obtained according to any of the methods described herein is also provided. The present disclosure relates to methods and compositions for exogenous sequence integration, including parallel (simultaneous) or sequential exogenous sequence integration (including transgene stacking) in a plant species, including in a polyploid plant. In yet another aspect, disclosed herein is a plant, plant part, seed, or fruit comprising one or more plant cells comprising a targeted genomic modification to one or more alleles of an endogenous gene in the plant cell, wherein the genomic modification follows cleavage by a site specific nuclease, and wherein the genomic modification produces a mutation in the endogenous gene such that the endogenous gene produces a product that results in an herbicide tolerant plant cell. In yet another aspect, disclosed herein is a method for making a plant cell as disclosed herein above, the method comprising: expressing one or more site specific nucleases in the plant cell; and, modifying one or more alleles of an endogenous gene across multiple genomes of a polyploid plant cell. In another aspect, provided herein is a seed from a plant comprising the plant cell that is obtained as described her
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