Detection method for CRISPR-Cas9 edited tomato with single nucleotide deletion using multiplex probe-based real-time PCR and confirmation of non-transgenicity: A case study

The adoption of gene-edited (GE) food crops has increased due to applicability of technology without drastically affecting the genetic information. However, the discussion on regulatory regime pertaining to GE plants throughout the globe is overwhelming. In the countries where GE products are regulated, or for the verification of gene-editing as claimed by the developers, detection of GE plants (referred to as GE detection here) plays an imperative role. Precisely detecting small modifications in single or few nucleotide(s) is a challenge. An efficient detection method was developed for GE tomato line with a single base pair edit as deletion in Solanum lycopersicum pectate lyase (SlPL) gene for better shelf life. A stepwise strategy is reported herein, comprising of screening at early phase targeting Cas9 protein gene using rapid loop-mediated isothermal amplification (LAMP) and conventional polymerase chain reaction (PCR) assays, followed by the verification of single point mutation using real-time PCR. Multiplex TaqMan® real-time PCR using fluorescent labelled dual probes simultaneously targeting edited and unedited sequences, was used for verification of single point deletion. This method was based on the negative selection where the presence of a mutation was determined by absence of signal in comparison to the wild-type. The developed multiplex real-time PCR method was found sensitive enough to detect 0.1% of the targeted lines. In view of the regulation of GM tomato in several countries, the approach for confirming non-transgenic nature of edited line was also presented using real-time PCR targeting common screening elements present in globally approved GM tomato events. This approach could be utilized as a tool globally either to support the regulatory authorities for food traceability or to verify the editing as well as to track suspected transgenic element in SDN-1 and SDN-2 types.