Systematic Analysis of the Effect of Genomic Knock-Out of Non-Essential Promiscuous HAD-Like Phosphatases YcsE, YitU and YwtE on Flavin and Adenylate Content in Bacillus Subtilis.

Studying the metabolic role of non-essential promiscuous enzymes is a challenging task, as genetic manipulations usually do not reveal at which point(s) of the metabolic network the enzymatic activity of such protein is beneficial for the organism. Each of the HAD-like phosphatases YcsE, YitU and YwtE of Bacillus subtilis catalyzes the dephosphorylation of 5-amino-6-ribitylamino-uracil 5'-phosphate, which is essential in the biosynthesis of riboflavin. Using CRISPR technology, we have found that the deletion of these genes, individually or in all possible combinations failed to cause riboflavin auxotrophy and did not result in significant growth changes. Analysis of flavin and adenylate content in B. subtilis knockout mutants showed that (i) there must be one or several still unidentified phosphatases that can replace the deleted proteins; (ii) such replacements, however, cannot fully restore the intracellular content of any of three flavins studied (riboflavin, FMN, FAD); (iii) whereas bacterial fitness was not significantly compromised by mutations, the intracellular balance of flavins and adenylates did show some significant changes.

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

To prevent food fraud, products can be monitored by various chemical-analytical techniques. In this study, we present a CRISPR-Cpf1 DETECTR-based assay for the differentiation of plant ingredients in sweet confectionary like fine and bulk-cocoa, or bitter and sweet almonds. To enable rapid in-field analysis, the trans-cleavage activity of the Cpf1 enzyme was used to develop a DETECTR (DNA endonuclease-targeted CRISPR trans reporter) assay for simple, highly specific fluorometric detection of single nucleotide polymorphisms (SNPs). The endonuclease Cpf1 requires the protospacer adjacent motif (PAM) 5'-TTTV-3' for activation, but the recognition sequence is freely programmable. The SNPs were selected to alter the Cpf1 specific PAM sequence. As a result, sequences that do not carry the canonical PAM sequence are not detected and thus not cut. The optimized system was used for both raw material and processed products such as cocoa masses or marzipan with a limit of detection of 3 ng template DNA. In addition, we were able to implement the system in the context of an LFA (lateral flow assay) to serve as a basis for the development of rapid test systems. Supplementary Information: The online version contains supplementary material available at 10.1007/s12161-023-02500-w.

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.