Recent advances and applications in integrating nucleic acid amplification technologies and CRISPR systems by spatial / temporal separation strategies for one-pot diagnosis

Abstract

The integration of nucleic acid amplification techniques (NAATs) with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems has enabled a new generation of molecular diagnosis that combines high sensitivity with single-nucleotide specificity. More importantly, consolidating both processes into a unified and airtight “one-pot” format without “non-closed” reagent transfer steps holds substantial promise for point-of-care testing (POCT) in resource-limited settings, particularly in clinical situations requiring rapid responses, such as during pandemics. This approach offers streamlined workflows, faster turnaround times, and reduced risk of contamination. However, the intrinsic biochemical incompatibility between amplification enzymes and CRISPR nucleases, as the former typically generate more nucleic acids while the latter consumes them to produce a signal, particularly due to competing reaction kinetics, poses a major challenge for one-pot assay development. In recent years, significant progress has been made in addressing this issue through a range of biochemical and engineering strategies. This review systematically categorizes these strategies based on whether NAATs and CRISPR systems are fully separated at the onset of the reaction, classifying them into spatial separation and temporal separation approaches. We highlight key representative studies within each category and evaluate their underlying mechanisms, performance trade-offs, and practical limitations. Furthermore, we discuss the remaining challenges that hinder clinical translation and provide insights into future directions, such as integration with upstream sample preparation and downstream signal readout modules. We hope this review not only provides a coherent roadmap for the rational design of one-pot platforms but also inspires broader innovation at the interface of molecular engineering in real-world healthcare applications.