Case Studies in the Molecular Characterization of Genome-Edited Soybean and Tomato

Genome editing (GE) technologies such as CRISPR-Cas systems enable one or a few precise, targeted genetic modifications to achieve specific beneficial outcomes, compared with the disparate, numerous, untracked, and random genetic modifications that arise through conventional breeding. While most GE products are fundamentally similar to conventionally bred varieties, thorough molecular characterization may still be required to support regulatory submissions in countries that mandate such assessments. Using two Syngenta GE lines – E001SYFT soybean and E001TMFM tomato – we evaluated how Illumina® short-read whole genome sequencing (WGS) can address molecular endpoints that might be relevant for product deregulation in some jurisdictions, while highlighting fit-for-purpose alternatives where appropriate. WGS of GE products was used to effectively address three endpoints that may be requested by regulatory agencies: (1) Target gene edit characterization confirmed precise edits identical to those verified by other methods such as PCR followed by Sanger sequencing; (2) Empirical evaluation of off-target sites confirmed the absence of modifications at predicted sites demonstrating the high specificity of the CRISPR-Cas12a system and supporting the conclusion that in silico analysis is sufficient to assess this endpoint; (3) Assessment of no exogenous DNA edited line (NEDEL) status, which confirms the absence of transformation vector sequences. Alternatively, NEDEL status may be confirmed via high resolution targeted sequencing using hybridization capture followed by Next Generation Sequencing . The high-resolution data generated through Illumina® short-read WGS provides a robust analytical path to satisfy product characterization needs when such requirements are established. Therefore, when dictated by existing regulations, we advocate for promoting efficient, science-based approaches to product deregulation that focus on targeted, endpoint-specific data analyses. While WGS offers a comprehensive analytical option, these endpoints can also be effectively addressed through various fit-for-purpose and targeted molecular techniques.