One-pot diagnostic methods based on CRISPR/Cas and Argonaute nucleases: strategies and perspectives.

Programmable nuclease-based detection technology has superior sensitivity, specificity, portability, and timeliness. Interference between amplification and programmable nucleases presents a major barrier to the clinical application of programmable nuclease-based diagnosis. A one-pot assay combining amplification and nuclease cleavage could overcome issues of contamination and operation complexity. Strategies encompassing physical isolation, microfluidic chips, thermal activation, component optimization, nuclease characterization and engineering, and guide RNA modification for the development of one-pot detection are reviewed. Existing one-pot detection methods still have room for improvement, which will be necessary to promote the popularization and application of programmable nuclease-based detection technology. CRISPR/Cas and Argonaute (Ago) proteins, which target specific nucleic acid sequences, can be applied as diagnostic tools. Despite high specificity and efficiency, achieving sensitive detection often necessitates a preamplification step that involves opening the lid and multistep operation, which may elevate the risk of contamination and prove inadequate for point-of-care testing. Hence, various one-pot detection strategies have been developed that enable preamplification and sensing in a single operation. We outline the challenges of one-pot detection with Cas and Ago proteins, present several main implementation strategies, and discuss future prospects. This review offers comprehensive insights into this vital field and explores potential improvements to detection methods that will be beneficial for human health. [ABSTRACT FROM AUTHOR]