Literature review of baseline information to support the risk assessment of RNAi‐based GM plants

Abstract

This report is the outcome of an EFSA procurement aiming at investigating and summarising the state of knowledge on (I) the mode-of-action of dsRNA and miRNA pathways, (II) the potential for nontarget gene regulation by dsRNA-derived siRNAs or miRNAs, (III) the determination of siRNA pools in plant tissues and the importance of individual siRNAs for silencing. The report is based on a comprehensive and systematic literature search, starting with the identification and retrieval of~190,000 publications related to the research area and further filtered down with keywords to produce focused collections used for subsequent screening of titles and abstracts. The report is comprised of an (I) Introduction to the field of small RNAs, (II) a Data and Methodologies section containing strategies used for literature search and study selection, and (III) the Results of the literature review organized according to the three main procurement tasks. The outcome of the first task reviews dsRNA and miRNA pathways in mammals (including humans), birds, fish, arthropods, annelids, molluscs, nematodes, and plants. Eight taxon-dedicated chapters are based on~1,400 cumulative references chosen from~10,000 inspected titles and abstracts. We review conserved and divergent aspects of small RNA pathways and dsRNA responses in animals and plants including structure and function of key proteins as well as four basic mechanisms: genome-encoded posttranscriptional regulations (miRNA), degradation of RNAs by short interfering RNA pools generated from long dsRNA (RNAi), transcriptional silencing, and sequence-independent responses to dsRNA. The outcome of the second task focuses on base pairing between small RNAs and their target RNAs and predictability of biological effects of small RNAs in animals and plants. The outcome of the last task reviews methodology, siRNA pools, and movement of small RNAs in plants. Potential transfer of small RNAs between species and circulating miRNAs in mammals is described in the final chapter.

Risk Assessment Considerations for Genetically Modified RNAi Plants: EFSA’s Activities and Perspective

“…The European Food Safety Authority has undertaken several activities on the risk assessment of RNAi-based GMPs to define in which areas existing risk assessment approaches for GMPs are suitable, or require complementary or alternative strategies. These include: (Pačes et al, 2017), while the FF safety report focused on the kinetics and possible effects of non-coding (nc) RNAs, including silencing RNAs, and upon ingestion by humans and animals (Dávalos et al, 2019). The report in support of the environmental risk assessment considered environmental RNAi-related aspects in arthropods, nematodes, and annelids and molluscs (Christiaens et al, 2018).…”

“…Based on the available knowledge, EFSA's GMO Panel (see text footnote 1) considers that for plants a group of in silico parameters enables the prediction of offtargets, while for human and animals the available tools may not allow for sufficiently reliable predictions (Pinzón et al, 2017). Bioinformatic analyses for off-targets is based on several criteria (e.g., degree and position of base-pairing between the small RNA and transcript) that determine the efficiency of silencing (reviewed by Pačes et al, 2017). Therefore, in silico target prediction algorithms are designed based on criteria related to the biochemical and thermodynamical properties of base pairing, among other filtering parameters (Rhoades et al, 2002;Pasquinelli, 2012).…”

“…Therefore, in silico target prediction algorithms are designed based on criteria related to the biochemical and thermodynamical 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 whether a dsRNA or artificial miRNA is used, a heterogeneous pool of siRNAs versus a more homogeneous pool of miRNAs will be produced, impacting the silencing of the potential off-target gene (Pačes et al, 2017).…”

RNA‐based biocontrol compounds: current status and perspectives to reach the market

“…FIFRA responded to this document by organizing a meeting the following year that explicitly addressed several concerns pertaining to ecological risk considerations and human health considerations, and provided various recommendations to address uncertainties related to these concerns . A similar exercise was conducted by the European Food Safety Authority (EFSA) which triggered the collection of literature reviews based on scientific information from RNAi studies . These reviews, intended to support the risk assessment of RNAi‐based GM plants, provide a comprehensive overview of RNAi and will also be useful in the development of regulatory guidelines for externally applied RNA‐based biocontrol compounds.…”

“…36 A similar exercise was conducted by the European Food Safety Authority (EFSA) which triggered the collection of literature reviews based on scientific information from RNAi studies. [37][38][39] These reviews, intended to support the risk assessment of RNAi-based GM plants, provide a comprehensive overview of RNAi and will also be useful in the development of regulatory guidelines for externally applied RNA-based biocontrol compounds. With a similar objective, an Organisation for Economic Co-operation and Development (OECD) conference on RNA-based pesticides was recently organized in April 2019 in Paris, with the aim of providing an overview of the current status and future possibilities for the regulation of externally applied dsRNA-based compounds.…”