A comparative analysis of insertional effects in genetically engineered plants

A comparative analysis of insertional effects in genetically engineered plants: considerations for pre-market assessments
Jaimie Schnell , Marina Steele , Jordan Bean , Margaret Neuspiel , Cécile Girard ,Nataliya Dormann , Cindy Pearson , Annie Savoie, Luc Bourbonnière3and Philip Macdonald

Abstract
During genetic engineering, DNA is inserted into a plant’s genome, and such insertions are often accompanied by the insertion of additional DNA, deletions and/or rearrangements. These genetic changes are collectively known as insertional effects, and they have the potential to give rise to unintended traits in plants. In addition, there are many other genetic changes that occur in plants both spontaneously and as a result of conventional breeding practices. Genetic changes similar to insertional effects occur in plants, namely as a result of the movement of transposable elements, the repair of double-strand breaks by non-homologous end-joining, and the intracellular transfer of organelle DNA. Based on this similarity, insertional effects should present a similar level of risk as these other genetic changes in plants, and it is within the context of these genetic changes that insertional effects must be considered. Increased familiarity with genetic engineering techniques and advances in molecular analysis techniques have provided us with a greater understanding of the nature and impact of genetic changes in plants, and this can be used to refine pre-market assessments of genetically engineered plants and food and feeds derived from genetically engineered plants.

Keywords Insertional effects Novel trait Unintended trait Genetic engineering Conventional breeding Pre-market assessment

@A comparative analysis of insertional effects in genetically engineered plants: considerations for pre-market assessments - Springer:

Transgenic ResearchAssociated with the International Society for Transgenic Technologies (ISTT)
© The Author(s) 201410.1007/s11248-014-9843-7

QUOTE:
Repair of double-strand [DNA] breaks by non-homologous end-joining

Double-strand breaks may be caused by physical stress on chromosomes, DNA replication across a nick, DNA crossing over during homologous recombination, or by reactive oxygen species, which are produced by the plant cell as a by-product of metabolism, in response to pathogen attack, or by exposure to environmental pollutants, ionizing radiation and ultraviolet light.
The main pathway for repair of double-strand breaks in plants is non-homologous end-joining (NHEJ). NHEJ is an error-prone recombination pathway, and deletions, insertions and rearrangements are often observed at the repair site where the two DNA ends have been rejoined (Gorbunova and Levy 1999). These are similar to the deletions, insertions and rearrangements that are often observed at the site of DNA insertion in plants having undergone genetic engineering. As with genetic engineering, if these deletions, insertions and rearrangements involve endogenous genes, the nature and/or regulation of these genes can be affected.
A hallmark of NHEJ repair sites is the presence of short repeats, sometimes referred to as microhomologies, that are typically 1–8 bp in length (Gorbunova and Levy 1999; Morita et al. 2009). These will be present when the repair is mediated by annealing of the exposed ends at these repeats. Interestingly, microhomologies are also often observed at sites where DNA has been inserted by genetic engineering, between the inserted DNA and the flanking plant genome sequences (Gorbunova and Levy 1999; Somers and Makarevitch 2004). This is because both Agrobacterium-mediated and particle bombardment transformation methods rely on the plant’s DNA repair pathways, in particular NHEJ, to introduce the DNA, although the exact mechanisms have not yet been fully elucidated (Makarevitch et al. 2003; Mayerhofer et al. 1991; Somers and Makarevitch 2004; Takano et al. 1997). Furthermore, the footprints created by excision of DNA transposons also contain these microhomologies because, once again, NHEJ is involved in repairing the double-strand break caused by excision (Huang and Dooner 2012; Rubin and Levy 1997). The common mechanism that underlies the generation of the insertions, deletions and rearrangements found at sites of DNA insertion via genetic engineering, double-strand break repair and DNA transposon excision is evidence that these insertional effects are not unique to genetic engineering....