This review article (purchase required) covers the recent application of CRISPR/Cas technology to food authenticity sensor applications.
The CRISPR/Cas system is a defence mechanism in bacteria and archaea against phage invasion. It allows them to store fragments of invading DNA and use them as a guide to recognize and destroy future invasions. Bound to a CRISPR RNA (crRNA) that precisely matches the nucleotide sequence of the target (spacer sequences), the CRISPR-associated (Cas) protein is precisely navigated to these sequences. Through enzymatic processes, it cleaves target nucleic acids at specific sites. This sequence selectivity underpins gene editing technology. It is less well known that it can also be used to build selective analytical sensors.
The authors describe how, in recent years, CRISPR/Cas-based detection systems have been applied to precise DNA detection for food authentication. Such systems entail designing a guide RNA that complements the target DNA, triggering precise cleavage by the Cas enzyme and activating collateral cleavage. This leads to the cleavage of fluorescent probes, enabling detection. CRISPR/Cas-based detection systems can be a rapid and highly specific tool for identifying target DNA in food. Coupled with DNA amplification strategies, it has the potential to achieve sub-attomolar sensitivity for nucleic acid detection.
CRISPR/Cas assays are qualitative, but can be made quantitative by combining with digital sample partitioning. The sample is divided into multiple individual partitions, each containing a distinct number of biological entities (0, 1, 2, 3, etc.). Each partition reacts independently and the partition containing the target will produce an increased fluorescent signal, allowing absolute quantification using Poisson distribution.
The authors cite and review published examples, and conclude that these digital isothermal amplifications and dCRISPR methods are rapid and offer single copy sensitivity and high accuracy, making them a promising tool for accurate DNA quantification with great potential for application in authenticating food DNA.
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