High-throughput targeted sequencing to detect gene-edited plants

Similar to genetically modified organisms (GMO) produced by classical genetic engineering, gene-edited organisms and derived food/feed products commercialized on the European market fall within the scope of the European Union Directive 2001/18/EC. Consequently, their control in the food/feed chain by GMO enforcement laboratories is required by the competent authorities to guarantee food/feed safety and traceability (2003/1829/EC; 2003/1830/EC). However, their detection is potentially challenging both at the analytical and interpretation levels, since it requires methodological approaches that can target and detect a specific single variation point introduced into a gene-edited organism. In this study, a targeted high-throughput sequencing approach was proposed. This approach includes a prior PCR-based enrichment step to amplify regions of interest followed by a subsequent data analysis methodology to identify single variation points of interest . To investigate if the performance of this targeted sequencing approach is compatible with the criteria used in the GMO detection field, several samples, containing different percentages of a gene-edited rice line carrying a single adenosine nucleotide insertion in OsMADS26, were prepared and analysed. The single variation point of interest in samples containing the gene-edited rice line could successfully be detected, both at high and low percentages. No impact related to food processing nor to the presence of other crop species was observed. The proposed targeted sequencing approach consequently constitutes as a specific and sensitive tool that can be helpful to support the safety and traceability of the food/feed chain regarding gene-edited plants carrying single variation points.