Targeted high-throughput sequencing enables the detection of single nucleotide variations in CRISPR/Cas9 gene-edited organisms

Fraiture Marie-Alice, D’aes Jolien, Guiderdoni Emmanuel, Meunier Anne Cecile, Delcourt Thomas, Hoffman Stefan, Vandermassen Els, De Keersmaecker Sigrid C. J., Vanneste Kevin, Roosens Nancy. 2023. Targeted high-throughput sequencing enables the detection of single nucleotide variations in CRISPR/Cas9 gene-edited organisms. Foods, 12 (3) : 1-14.

https://doi.org/10.3390/foods12030455

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Résumé : Similar to genetically modified organisms (GMOs) produced by classical genetic engineering, gene-edited (GE) organisms and their derived food/feed products commercialized on the European Union market fall within the scope of 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 at both the analytical and interpretation levels since this requires methodological approaches that can target and detect a specific single nucleotide variation (SNV) introduced into a GE organism. In this study, we propose a targeted high-throughput sequencing approach, including (i) a prior PCR-based enrichment step to amplify regions of interest, (ii) a sequencing step, and (iii) a data analysis methodology to identify SNVs of interest. To investigate if the performance of this targeted high-throughput sequencing approach is compatible with the performance criteria used in the GMO detection field, several samples containing different percentages of a GE rice line carrying a single adenosine insertion in OsMADS26 were prepared and analyzed. The SNV of interest in samples containing the GE rice line could successfully be detected, both at high and low percentages. No impact related to food processing or to the presence of other crop species was observed. The present proof-of-concept study has allowed us to deliver the first experimental-based evidence indicating that the proposed targeted high-throughput sequencing approach may constitute, in the future, a specific and sensitive tool to support the safety and traceability of the food/feed chain regarding GE plants carrying SNVs.

Mots-clés Agrovoc : génie génétique, Oryza sativa, séquence nucléotidique, organisme génétiquement modifié, variation génétique, technique analytique, adn

Mots-clés libres : Crispr/Cas9, Single nucleotide variations, Transgenic rice, Detection, PCR-based enrichment, High-throughput sequencing, Food and feed chain, Genetically modified organism

Classification Agris : Q01 - Sciences et technologies alimentaires - Considérations générales
Q04 - Composition des produits alimentaires
F30 - Génétique et amélioration des plantes

Champ stratégique Cirad : CTS 3 (2019-) - Systèmes alimentaires

Agences de financement hors UE : Belgian Federal Public Service of Health, Food Chain Safety

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