Negative Effects Of Gmos On The Environment – GMOs are relatively new, and like anything new, there are conflicting views on many issues related to the use of these plants. One area that is getting a lot of attention is whether these GMO plants and the foods that contain them are safe to eat. There is no data to indicate that consuming GMOs is bad for human health.

How do we know? GMOs have undergone more detailed evaluation than any other group of plants we eat. In almost all cases, GMOs differ from a conventional plant by adding only one or two genes that produce one or two new proteins. The origin and function of these proteins are well understood. For example, proteins are studied to ensure that they do not have characteristics that could cause allergic reactions. The National Academy of Sciences, the United States’ number one source for independent and objective advice to the nation on matters of science and technology, has concluded that GMOs are safe for human health. A large number of prestigious health and scientific organizations around the world have come to the same conclusion. Furthermore, in the two decades that GMOs have been on the market, there have been no health problems due to genetically modified organisms.

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As GMOs currently stand, there are no health benefits to eating them over non-GMO foods. However, this may change in the future as technology develops and becomes more sophisticated. Genetically modified (GMP) plants intended for market release can be engineered to induce “gene silencing” through RNA interference (RNAi). The European Food Safety Authority (EFSA) and other international risk assessment bodies/regulatory agencies have undertaken several actions to determine whether existing risk assessment approaches for GMPs are appropriate for RNAi-based GMP risk assessment or require complementary or alternative approaches. To our knowledge, no dedicated guidelines for the risk assessment and regulation of RNAi-based GMPs have been developed internationally, confirming that existing science-based risk assessment approaches for GMPs are generally considered appropriate for GMP- RNAi-based assays. However, some peculiarities have been identified for the risk assessment of RNAi-based GMPs. Here, we report some of these specificities identified and addressed by the EFSA Panel on GMOs for molecular characterization, food/feed safety assessment and environmental risk assessment of RNAi-based GMPs, using maize MON87411 expressing DvSnf7 dsRNA as a study of case. .

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Genetically modified plants (GMP) and/or derived food/feed products (FF) are subject to risk assessment and regulatory approval before entering the market in the European Union (EU). In this process, the role of the European Food Safety Authority (EFSA) is to assess and provide scientific advice to risk managers on any possible risks that the implementation (e.g. consumption or cultivation) of GMPs may present to people, animals and the environment. (Waigmann et al., 2012). EFSA’s scientific advice on the risk assessment of GMPs is provided by its Scientific Panel on Genetically Modified Organisms (GMOs) made up of scientific experts drawn from EU research institutes, universities or risk assessment bodies. For the assessment of GMP market registration applications, EFSA’s GMO Panel is supported by the GMO Unit and three permanent working groups, each of which focuses on specific areas of risk assessment addressing: (a) molecular characterization of GMPs; (b) FF safety assessment of GMP and/or derived FF products; and (c) environmental risk assessment of GMPs (see Figure 1 for further details; Devos et al., 2014).

Figure 1. Risk assessment approach for genetically modified plants [reprinted with permission from the EFSA infographic (available at https://www.efsa.europa.eu/en/discover/infographics/risk-assessment-genetical-modified-plants; ISBN 978 ) -92-9199-913-2 | two: 10.2805/240762 | TM-02-17-009-EN-N)].

Plants can be engineered to induce gene silencing by RNA interference (RNAi). Currently, RNAi-based GMPs have been designed to express either a double-stranded RNA (dsRNA) precursor or an artificial microRNA (miRNA) precursor. These molecules are cleaved by Dicer/Dicer-like proteins into a group of small RNAs, 20–30 nucleotides in length (small interfering RNAs [siRNAs] or miRNAs) that specifically bind RNA target/messenger (mRNA) with perfect or near. perfect complementarity (Burand and Hunter, 2013; Koch and Kogel, 2014; Cagliari et al., 2019). siRNAs and miRNAs bind to an Argonaute protein forming the RNAi-induced silencing complex that, based on sequence homology, targets cognate RNAs. Current RNAi-based GMPs typically express a dsRNA that is designed to either down-regulate an endogenous plant mRNA (eg, to alter nutrient composition) or a gene in pests or pathogens that infest these plants , so-called environmental RNAi (eg, Ivashuta). et al., 2015).

Small interfering RNAs and miRNAs can also trigger gene silencing in the plant other than their intended targets (i.e., off-targets), giving rise to unintended phenotypes (Casacuberta et al., 2015).

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The European Food Safety Authority has undertaken several activities on RNAi-based GMP risk assessment to define in which areas existing GMP risk assessment approaches are appropriate or require complementary or alternative strategies. These include:

1. International Scientific Workshop “Risk Assessment Considerations for RNAi-Based Genetically Modified Plants” (4-5 June 2014, Brussels, Belgium: at this workshop, academic experts, risk assessment bodies, non-governmental organizations, the European Commission and private sector institutions identified scientific uncertainties regarding the level of exposure of humans, animals and the environment to artificial dsRNA/miRNA and derived small RNAs, hereafter referred to as silencing RNAs, as well as limitations of in silico methods to identify unequivocally potential non-targets ( European Food Safety Authority [EFSA], 2014).

2. External scientific reports: EFSA commissioned three external scientific reports in which the relevant scientific literature was systematically reviewed to further inform the molecular characterization, FF safety assessment and environmental risk assessment of RNAi-based GMPs and to address issues identified in the workshop. The report supporting the molecular characterization addressed the dsRNA and miRNA pathways in different species, including mammals, arthropods and plants (Pačes et al., 2017), while the FF safety report focused on the kinetics and possible effects of non-coding RNAs ( nc). , including attenuation of RNAs and upon ingestion by humans and animals (Dávalos et al., 2019). The report in support of environmental risk assessment considered aspects of environmental RNAi in arthropods, nematodes and annelids and molluscs (Christiaens et al., 2018).

3. Internal note on off-target risk assessment and prediction strategy: In 2017, EFSA’s GMO Panel published an internal note

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On the strategy to identify/predict off-targets and risk assessment of their potential impact in RNAi-based GMPs. It has been built on the basis of available scientific knowledge and is expected to evolve with the advancement of knowledge in the field.

4. Opinions of the GMO panel of the RNAi-based GMPs: EFSA’s GMO panel assessed applications for market registration for the import and processing for food and feed uses of potato EH92-527-1 (including cultivation in the EU) and soybean MON87705, 305423, MON87705 × MON89788, and 305423 × 40−3−2 (excluding cultivar) designed to down-regulate endogenous plant transcripts that modulate potato tuber amylose and starch content or grain fatty acid profile soybean (EFSA Panel on Genetically Modified Organisms [EFSA GMO], 20012a), 2015; EFSA Panel on Genetically Modified Organisms [EFSA GMO], 2013, 2016, respectively). More recently, the GMO panel also assessed the maize events MON87411 and MON87427 × MON89034 × MIR162 × MON87411 that constitute cases of environmental RNAi (EFSA Panel on Genetically Modified Organisms [EFSA GMO], Naegeli et al., 2018, 2019, respectively) . Maize MON87411 expresses, among others, an insecticidal DvSnf7 dsRNA that down-regulates Snf7 transcription in the western corn rootworm (Diabrotica spp.) and confers protection against this major corn pest. Some aspects of risk assessment in MON87411 maize are discussed below.

Table 1. Overview of the activities of the European Food Safety Authority on the risk assessment of genetically modified plants with RNA interference.

The specificity of RNA interference is based on the sequence identity between silent small RNAs and mRNA targets; however, other transcripts with sufficient sequence identity to small silencing RNAs may also be targeted for destruction leading to off-target effects (European Food Safety Authority [EFSA], 2014; Federal Law on Insecticides, Fungicides and Rodenticides [FIFRA], Scientific Advisory Panel). [SAP], 2014; Ramón et al., 2014; Casacuberta et al., 2015). Thus, identifying off-targets would facilitate risk assessment. Off-targets could occur in GMP itself or in other organisms that are exposed to GMP and derived products through consumption. Based on available knowledge, EFSA’s GMO panel (see text footnote 1) considers that, for plants, a set of in silico parameters allows the prediction of off-targets, while for humans and animals the available tools it might not allow sufficiently reliable predictions (Pinzón et al., 2017). Bioinformatic analyzes for off-targets are based on several criteria (eg, the degree and position of base pairing between the small RNA and the transcript) that determine silencing efficiency (reviewed by Pačes et al., 2017). Therefore, in silico target prediction algorithms are designed based on criteria related to biochemical and thermodynamic properties of base pairing, among other filtering parameters (Rhoades et al., 2002; Pasquinelli, 2012). In addition, other factors that can impact these interactions and lead to off-targets is the abundance of each small RNA produced (Pačes et al., 2017). Depending on the use of an artificial dsRNA or miRNA, a heterogeneous group of siRNAs versus a more homogeneous group of miRNAs will be produced, which will impact the silencing of

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