Genetic authentication: Differentiation of fine and bulk cocoa (Theobroma cacao L.) by a new CRISPR/Cas9-based in vitro method

Abstract

Ecuador is one of the most important producers of fine cocoa. In addition to the fine cocoa type Nacional (Arriba), however, the cheaper but less aromatic bulk cocoa “Colección Castro Naranjal 51” (CCN-51) is also cultivated there. In a previous work, several characteristic SNPs could be identified to distinguish these two varieties. However, the detection of single base exchanges in order to differentiate raw material types or varieties is challenging, as some of the methods require the use of restriction endonucleases. As a prerequisite, the application is only successful if an appropriate recognition site is available. The use of the programmable Cas9 endonuclease known from the genome editing system CRISPR/Cas9 is a powerful alternative. For the detection of bulk cocoa in fine cocoa a single nucleotide polymorphism (SNP) was selected, which is located within a PAM region mandatory for the Cas9 endonuclease. Consequently, only the bulk cocoa is attacked by the nuclease. The result can be recorded using agarose or capillary gel electrophoresis (AGE and CGE). Both methods yielded comparable results. AGE can be used for a semi-quantitative estimation and the more sophisticated CGE for quantitation based on a calibration line. A reliable detection could be made up to an admixture of 10% CCN-51 which should represent a realistic scale for routine applications.