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

Abstract

Cocoa cultivation is dominated by the clone "Collecioń 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.

Capillary electromigration methods for food analysis and Foodomics: Advances and applications in the period March 2021 to March 2023

“…The putative candidate genes identified in this work could be useful for enhancing the shelf life of dessert-type cantaloupe melons. CE methodologies applied to DNA analysis reported in the last 2 years have been aimed at assessing food safety [57][58][59], detecting genetically modified organisms (GMOs) [60,61], evaluating food authenticity [62,63], and detecting food fraud [64,65].…”

“…CE methodologies applied to DNA analysis reported in the last 2 years have been aimed at assessing food safety [57–59], detecting genetically modified organisms (GMOs) [60, 61], evaluating food authenticity [62, 63], and detecting food fraud [64, 65].…”

“…Finally, La‐Rostami et al. proposed a rapid methodology to distinguish between two cocoa varieties (CCN‐51 and Arriba ) of different quality with the aim of preventing food fraud [64]. They optimized an in vitro clustered regularly interspaced short palindromic repeats assay (CRISPR‐Cpf1) assay for the specific detection of single nucleotide polymorphism in cocoa.…”

“…The results obtained when the developed PCR-CE approach was applied to 40 commercial food products pointed out their potential for the simultaneous analysis of multiple cod species, which may have a special interest for the fish industry. Finally, La-Rostami et al proposed a rapid methodology to distinguish between two cocoa varieties (CCN-51 and Arriba) of different quality with the aim of preventing food fraud [64]. They optimized an in vitro clustered regularly interspaced short palindromic repeats assay (CRISPR-Cpf1) assay for the specific detection of single nucleotide polymorphism in cocoa.…”

Variety Differentiation: Development of a CRISPR DETECTR Method for the Detection of Single Nucleotide Polymorphisms (SNPs) in Cacao (Theobroma cacao) and Almonds (Prunus dulcis)

“…However, using Cas9 from Streptococcus pyogenes only one suitable SNP was available for differentiation by CRISPR-Cas9 technology (Herrmann et al 2014 ). To obtain more available targets, we previously used a CRISPR-Cpf1 (from Prevotella and Francisella ) (Jinek et al 2012 ) based approach, which increased available detection sites on the AT-rich plastid genome in T. cocoa (La-Rostami et al 2022 ).…”