Impact of differential DNA methylation on transgene expression in cotton (Gossypium hirsutum L.) events generated by targeted sequence insertion.

Targeted Genome Optimization (TGO) using site-specific nucleases to introduce a DNA double-strand break (DSB) at a specific target locus has broadened the options available to breeders for generation and combination of multiple traits. The use of targeted DNA cleavage in combination with homologous recombination (HR)-mediated repair, enabled the precise targeted insertion of additional trait genes (2mepsps, hppd, axmi115) at a pre-existing transgenic locus in cotton. Here we describe the expression and epigenome analyses of cotton Targeted Sequence Insertion (TSI) events over generations.

Directed genome engineering for genome optimization.

The ability to develop nucleases with tailor-made activities for targeted DNA double-strand break induction at will at any desired position in the genome has been a major breakthrough to make targeted genome optimization feasible in plants. The development of site specific nucleases for precise genome modification has expanded the repertoire of tools for the development and optimization of traits, already including mutation breeding, molecular breeding and transgenesis.Through directed genome engineering technology, the huge amount of information provided by genomics and systems biology can now more effectively be used for the creation of plants with improved or new traits, and for the dissection of gene functions.

Targeted molecular trait stacking in cotton through targeted double-strand break induction.

Recent developments of tools for targeted genome modification have led to new concepts in how multiple traits can be combined. Targeted genome modification is based on the use of nucleases with tailor-made specificities to introduce a DNA double-strand break (DSB) at specific target loci. A re-engineered meganuclease was designed for specific cleavage of an endogenous target sequence adjacent to a transgenic insect control locus in cotton.

Homologous recombination: a basis for targeted genome optimization in crop species such as maize.

In an attempt to understand the feasibility of future targeted genome optimization in agronomic crops, we tested the efficiency of homologous recombination-mediated sequence insertion upon induction of a targeted DNA double-strand break at the desired integration site in maize. By the development of an efficient tissue culture protocol, and with the use of an I-SceI gene optimized for expression in maize, large numbers of precisely engineered maize events were produced in which DNA integration occurred very accurately. In a subset of events examined in detail, no additional deletions and/or insertions of short filler DNA at the integration site were observed.

Spontaneous mutation frequency in plants obscures the effect of chimeraplasty.

In this study we tested the performance of chimeraplasts, chimeric RNA/DNA oligonucleotides, for the creation of directed changes in chromosomal sequences in tobacco and oilseed rape. As target genes for chimeraplasty, the endogenous als gene and two transgenes, bar and a fusion between egfp and bar, were used. In experiments in which similar numbers of cells were treated with and without chimeraplasts, delivery of chimeraplasts did not lead to increased numbers of herbicide-resistant or egfp fluorescent calli.