Korean scientists used CRISPR-Cas to try and remove a section of DNA important for cold tolerance from the genome of the plant species Arabidopsis thaliana. The Crispr-Cas9 tool was used to simultaneously target and silence three genes in the genome. The three genes are very similar in their structure and located very close together in the genome. Such structures are called a 'gene cluster'.
It was shown that gene editing outcomes do not solely depend on the structure of the DNA. Epigenetics can also be a decisive factor i.e. gene regulation. In short, epigenetic mechanisms can be decisive for which genes in the cells are activated and which are silenced. It is as if the genome is the script, the cells are the actors and epigenetics take the part of the director who decides which actor speaks which text. One important factor is the way in which the chromosomes are packed: Gene activity is lower in areas where they are tightly packed. The experiment was carried out as basic research.
What is the problem?
Several publications show increasing evidence that gene regulation must be taken into account in assessing the outcomes and risks of genetic engineering. Indeed, the results from the experiments with the Arabidopsis plants were surprising: Three so-called 'lines' of the same species were used; all had different origins. They all had the same gene sequences in regard to cold tolerance. However, while the success rate of the intended gene manipulation in one line of Arabidopsis was 33 percent, in another line it was only 3.7 percent i.e. only about a tenth of the former. According to the authors, epigenetic effects were likely to be responsible for the differences between the different lines.
One plausible explanation: To enable the gene scissor to ‘cut’ its target site, the strands of DNA first have to be opened. If the strands of DNA are tightly packed in this region, the nuclease cannot or not efficiently do its job. One could say that, depending on origin, specific sites in the genome are 'protected' to a higher or lower degree against changes in the structure of the DNA.
Currently, differences in gene regulation are not taken into account in the risk assessment of genetically engineered plants. However, such differences can become much more of a risk if, for example, environmental conditions are affected due to climate change, or if genetically engineered organisms introgress the genome of wild relatives. Under such circumstances, the genomic changes can trigger adverse effects even if no such effects are observed in the laboratory or in field trials.
The Arabidopsis plants showed a change in their genomes that could hardly have been achieved with other methods: Clusters of genes containing multiple sequences of identical genetic information are frequently found in plant genomes. They are part of evolutionary mechanisms whereby duplications of DNA sequences can function as backup copies of redundant genetic information and/or enhance specific gene functions.
It is very difficult to change the biological function of gene clusters through e.g. random mutation because, in most cases, these changes will only affect single gene sequences and not all copies of the respective gene sequences. However, the gene scissor CRISPR/Cas9 works with the help of small guide-RNA, which acts as a tiny signpost to the target site in the genome. The gene scissor works in parallel on all the locations in the genome where DNA is found with the same structure for which the guide-RNA is programmed.
If the application of CRISPR/Cas is successful, the relevant natural genetic information is completely removed from the plants, even if several copies of the DNA sequences were originally present in the genome. Therefore, even if no additional DNA is inserted, this kind of genetic manipulation is significantly different to methods used in conventional breeding. Thus, the risks of such genetic manipulations need to be thoroughly assessed in detail.