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.

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.

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.