Integrated one-pot CRISPR-Cas detection: Addressing strategies and future prospects

Abstract

Integrated one-pot CRISPR-Cas detection combines isothermal amplification with CRISPR-Cas-mediated recognition in a single vessel, streamlining workflows, reducing time, and minimizing contamination risks for point-of-care diagnostics. This review systematically outlines current advances and core challenges, focusing on strategies to enhance performance. Existing methods achieve temporal and spatial coordination of amplification and detection through physical compartmentalization, chemical regulation, or enzymatic modulation, effectively mitigating interference between Cas enzymes and amplification systems to boost sensitivity and reliability. Despite progress, issues remain in system compatibility, multiplexing, sample matrix interference, and portable quantitative readouts. Future progress will rely on protein engineering for optimized Cas variants, artificial intelligence-assisted crRNA design, fully integrated microfluidic devices with lyophilized reagents, and non-optical detection modalities such as electrochemical or colorimetric.