The new plant breeding techniques provide the emergence of novel products that challenges our current regulations and our management practices of what we traditionally have viewed as a genetically modified organism (GMO). International regulations, such as the Cartagena Protocol on Biosafety, operate with definitions of GMOs that may not be applicable to products arising from some of these new techniques. The question then arises on how society and regulatory bodies should view and regulate the products. This report does not approach that problem per se, but as a crucial step in management, we sum up the current scientific understanding of two new plant breeding techniques, site directed nucleases (SDN) and oligonucleotide directed mutagenesis (ODM). The underlying mode of action of both of these techniques are the plants natural repair systems and how this can be utilized to achieve genomic modifications. Herein also lies the main challenge for risk assessment - our limited knowledge about the function of these systems, factors involved and potential off-target effects.
This report aims at providing an overview of the current status of scientific knowledge concerning SDN and ODM. We have reviewed up to date peer reviewed scientific publications on the mechanisms and natural functions that are utilized by SDN and ODM techniques in an effort to understand potential risks such as unintentional changes in the genome of plants. Finally, recommendations for action are outlined.
Site-direct nucleases are enzyme complexes that recognize specific DNA sequences in the genome and cleave them. The cleaved DNA is subsequently repaired by the organisms natural DNA repair systems. Currently, these enzymes are divided into four categories: meganucleases, zinc-finger, TALEN and CRISPR. The end product of the use of SDN techniques will largely depend on the design of the nuclease DNA recognition protein and the available template for repair. This report outlines potential unintended effects derived from the use of SDN that are mainly related to uncertainties regarding DNA target sequence specificity and DNA double-stranded break repair mechanism. There are several factors that influence both DNA binding and DNA repair, unfortunately they are to a large extent not fully understood. The lack of mechanistic understanding is a severe limitation for identifying potential hazards from SDNs and more research in this field is greatly recommended. Identifying unintentional effects in a system which is not fully understood becomes very difficult. However, using comprehensive untargeted profiling methods (such as omics) in order to detect and identify unintentional mutations with methodologies that are available today could minimize the potential hazards from SDN products.
Oligonucleotide-directed mutagenesis uses oligonucleotides to induce sequence specific mutations of native genomic sequences (i.e. genome editing). The introduced DNA is complementary to the genomic target sequence with the exception of a modification that usually is a deletion, insertion or a mismatch between the introduced synthesized DNA and the genomic DNA. There is scientific dispute on the DNA repair pathways and mechanisms that are induced, but briefly the cells DNA repair mechanisms detects the mismatch and repairs the genomic DNA using the introduced
modified DNA as a template. There are some reports on collateral and unspecific background mutations as side effects, but several research groups highlighted the uncertainties regarding those studies. Unintended effects that are described as induced by ODMs are related to cellular death, unpredicted mutations, mutation inheritance discrepancies, and others.
In conclusion, the two techniques reviewed in this report are not fully scientifically understood and thus poses many uncertainties connected to mode of action as well as potential unintentional effects. The safety assessment of such products should take into account risks associated with other existing user practices and habits, and the sources and nature of uncertainty that could not be addressed during the preceding steps of the risk assessment.
Finally, several recommendations have been proposed for the development of future research to contribute to the better understanding of the modes-of-action of such techniques and also to seek fulfilling the biosafety knowledge gaps. Overall, the results of this report indicate that biosafety considerations regarding new plant breeding technologies could, in principle, be addressed by the general approach developed for conducting the risk assessment of genetically modified crops. However, it is important to understand that the limitation that resides in the current understanding of these techniques regarding their potential adverse effects. Therefore, according to the requirements of a scientifically based risk assessment and the application of the precautionary principle, further biosafety research is highly recommended.
Objectives and scope
The aim of this report is to provide an assessment and identification of scientific knowledge gaps and uncertainties that are related to two new emerging technologies for plant breeding. In this context, new emerging technologies for plant breeding are mainly about changing the process of creating a new crop rather than changes in the traits carried by these organisms.
Within the site-directed nuclease-based (SDN) group of methods we have focused on the zinc finger technology as well as giving an overview of the other SDNs and oligonucleotide-directed mutagenesis (ODM) techniques. We have chosen not to discuss possible implementation of regulation concerning products resulting from the utilization of these plant-breeding methods.
Because commercialized products from ODM and SDN are not yet available in the market, it is not possible to address realistic risk scenarios, as well as case examples, that are connected to the choice of method used to generate modifications to the host plant genome. In addition, SDN and ODM are methods that promote the integration of foreign DNA or the modification of existing plant DNA. In any given plant modified by SDN or ODM techniques, the connected risk assessment must also take into account the transformation method and possibly the plant regeneration method as well as the inserted or modified DNA. This report does not address those tightly connected methods due to the lack of products available on the marked today resulting from SDNs or ODMs.
As mentioned above, there is a lot of knowledge concerning the mechanism or mode of action of the nucleases and repair mechanisms activated that needs to be uncovered. The purpose of this report is to show in what area these knowledge gaps reside. Therefore, we have reviewed scientific evidence including latest findings concerning unintentional changes in the genome of plants induced by these
techniques. Finally, the report provides insight into strategies for identification and management of these risks.
In addition, answers to the questions on the regulation of products from these new techniques, whether it falls within GMO legislation or not, were not within the scope of this report. However, this issue should be carefully taken into account not only to comply with national and international regulations but also to evaluate to what extent the precautionary principle should be invoked on products that might fall outside of existing safety regulations. The characteristics of these new crops and products may have adverse impacts in the environment, as well as socio-economic implications that have not yet been fully investigated.
The utilization of ODMs and SDNs is only one step in the generation of the final plant that will be grown in crop fields. Still there is the question of delivery of DNA/proteins to the cell and the generation of a plant from single cells. In addition crossing of the laboratory parental strain into elite crops raises the same issues as when generating elite crops from traditional genetic engineering, albeit with a much more difficult task of monitoring and detection.