Background and Objective

In the research project, potential biosafety risks are to be evaluated, which are to be examined from interactions, in particular between the gene sequences to be transferred (promoter, gene, terminator, cis and trans elements, etc.) and areas of the poplar genome. For example, the 35S promoter of the cauliflower mosaic virus, the promoter most frequently used for the regulation of genetically transferred genes (as well as other virally derived sequences), can interact with endogenous viruses that are "dormant" in the poplar genome, as well as with existing retroelements. These interactions could activate both the usually "dormant" retroelements/endogenous transposons and viruses, which can lead to increased, biosafety-relevant genomic instabilities, as well as completely new retroelements/transposons or viruses arising through recombination. The integration of the newly transferred genes in the poplar genome can also lead to inactivation or hyperactivation of endogenous genes.

In addition, the entire genome of poplars in which individual genes have been modified using the CRISPR/Cas9 system is to be sequenced and, if possible, "off-target". mutations are examined. Possible secondary effects of the genes mutated by genome editing are also conceivable.

Approach

In initiating experiments, already existing transgenic poplar lines were examined with regard to genomic stability using the AFLP technique. No genomic instabilities could be detected, neither the activation of "sleeping" retroelements/transposons nor of endogenous viruses. Next-generation sequencing methods are now being used to check genome modification methods (CRISPR/Cas) as a possible cause of genomic instability. No indications of significantly increased genomic instabilities as a result of the biotechnological methods compared to conventional mutagenesis could be found in any of the results obtained so far.

The methods of genome editing (CRISPR/Cas9, and others) work so precisely that (so far) only modifications could be found in the "desired" genes. There have not been any indications of "off-target" effects (so far). However, studies are also being carried out on nickase modifications in which no complete double-strand break can be observed, but only a single-strand cut in order to be able to exchange specific gene sections in a targeted manner. There are no results for this yet.

Preliminary Results

The results indicate that as expected a high genetic stability is existing between different transgenic lines and their respective non-transgenic controls. However, single cases have been observed where reproducible differences were detected in the AFLP pattern which indicate a transformation-induced genomic mutation. Further investigations are planned to unravel the basis for the observed variations.