Multiplexed CRISPR/Cas and Argonaute detection technology: strategies, challenges, and perspectives

An increasing number of applications demand the simultaneous detection of multiple nucleic acid targets within a single reaction, which results in a reduction in both the time required for the assay and the overall cost. The programmable nucleases Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) system and Argonaute have evolved into state-of-the-art molecular tools for the development of bioanalytical assays. In the review, the classification of the CRISPR/Cas and Argonaute were introduced followed by barriers of current multiplexed detection. Then the review systematically summarized the application of multiplexed CRISPR/Cas and Argonaute detection according to spatial/local separation, color-labelling differentiation, programmable nucleases sequence preference, sequence specific recognition. Finally, the review put forward the existing challenges and prospects of emerging field. The advancement of multiplexed CRISPR/Cas and Argonaute detection is ushering in a new era in bioanalysis with high sensitivity as well as specificity, offering great promise and desirability.