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 “Coleccion 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.

中文翻译：

---

基因验证：通过基于 CRISPR/Cas9 的新体外方法区分细粒和块状可可 (Theobroma cacao L.)

摘要 厄瓜多尔是优质可可最重要的生产国之一。然而，除了优质可可类型 Nacional (Arriba) 外，这里还种植价格便宜但芳香度较低的散装可可“Coleccion Castro Naranjal 51” (CCN-51)。在之前的工作中，可以识别出几个特征 SNP 来区分这两个品种。然而，为了区分原材料类型或品种而检测单碱基交换具有挑战性，因为某些方法需要使用限制性内切核酸酶。作为先决条件，只有在适当的识别站点可用时，申请才能成功。使用基因组编辑系统 CRISPR/Cas9 已知的可编程 Cas9 核酸内切酶是一种强大的替代方法。为了检测细可可中的大块可可，选择了单核苷酸多态性 (SNP)，它位于 Cas9 核酸内切酶必需的 PAM 区域内。因此，只有大块的可可会受到核酸酶的攻击。可以使用琼脂糖或毛细管凝胶电泳（AGE 和 CGE）记录结果。两种方法都产生了可比较的结果。AGE 可用于半定量估计，更复杂的 CGE 可用于基于校准线的定量。可靠的检测可以由 10% CCN-51 的混合物组成，这应该代表常规应用的现实规模。AGE 可用于半定量估计，更复杂的 CGE 可用于基于校准线的定量。可靠的检测可以由 10% CCN-51 的混合物组成，这应该代表常规应用的现实规模。AGE 可用于半定量估计，更复杂的 CGE 可用于基于校准线的定量。可靠的检测可以由 10% CCN-51 的混合物组成，这应该代表常规应用的现实规模。