In Vitro CRISPR-Cpf1 Assay for Differentiation of Fine and Bulk Cocoa ( Theobroma cacao L. ).

Cocoa cultivation is dominated by the clone "Colleción Castro Naranjal 51" (CCN-51). In contrast, CCN-51 is the expensive and aromatic fine cocoa "Arriba Nacional" from Ecuador. The differences in the overall quality of the beans and in the prices show that it is necessary to develop a rapid and accurate method to distinguish these varieties and prevent food fraud. To this end, we used a CRISPR-Cpf1 assay suitable for AT-rich targets such as the chloroplast genome (cpGenome). SNPs in cocoa plastid genomes were selected to replace the canonical PAM sequence of Cpf1 (5'-TTTV-3'). We developed two assay systems to digest both Arriba and CCN-51. The results were tested qualitatively by agarose gel electrophoresis and quantitatively by capillary gel electrophoresis. Using the assay described here, we were able to reliably detect admixtures of 5% CCN-51 ( P < 0.01) and 10% Arriba ( P < 0.05). The application to processed cocoa products was also successful.