Abstract
SummaryTargeted 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. In a subset of events, we observed variability in the level of transgene (hppd, axmi115) expression between independent but genetically identical TSI events. Transgene expression could also be differential within single events and variable over generations. This expression variability and silencing occurred independently of the transgene sequence and could be attributed to DNA methylation that was further linked to different DNA methylation mechanisms. The trigger(s) of transgene DNA methylation remains elusive but we hypothesize that targeted DSB induction and repair could be a potential trigger for DNA methylation.
Highlights
Targeted Sequence Insertion in cotton has been achieved by the introduction of a targeted DNA doublestrand break (DSB) and its repair by HRmediated insertion of trait genes
It has already been reported that targeted insertion events in tobacco, generated by Cre-lox mediated site-specific transgene integration into a specific chromosomal location can produce alleles that express at a predictable level, as well as alleles that are differentially silenced while the alleles were identical at the DNA sequence level
Co-delivery, using particle bombardment, of the COT-5/6 meganuclease gene and the respective donor DNAs into embryogenic callus (EC) of the target cotton line and consecutive selection of glyphosate tolerant EC events followed by PCR analysis allowed the recovery of targeted sequence insertion (TSI) events at frequencies ranging from 1.8 to 7.5% (Table S1)
Summary
Targeted Sequence Insertion in cotton has been achieved by the introduction of a targeted DNA DSB and its repair by HRmediated insertion of trait genes. By using a customized meganuclease, we were able to precisely insert two herbicide tolerance (HT) genes (2mepsps, hppd) in close vicinity to a preexisting transgenic locus in cotton and demonstrated that the resulting molecular stack was transmitted as a single locus to further generations (D’Halluin et al, 2013). We selected the transgene integration position with the objective to limit transcriptional interference between the transgenes of the molecular stack and to obtain more predictable expression of the newly added transgenes at the pre-existing good performing transgenic locus. It has already been reported that targeted insertion events in tobacco, generated by Cre-lox mediated site-specific transgene integration into a specific chromosomal location can produce alleles that express at a predictable level, as well as alleles that are differentially silenced while the alleles were identical at the DNA sequence level. Transcriptional gene silencing via DNA methylation was attributed as a trigger of the variation in transgene expression (Day et al, 2000)
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