Isothermal amplification and CRISPR/Cas one-pot detection systems: strategies and prospects

Molecular diagnostic techniques, known for their high sensitivity and specificity, are now the gold standard for detecting pathogen nucleic acids and are crucial in precision medicine. Traditional methodologies, including polymerase chain reaction (PCR) and gene sequencing, offer exceptional analytical precision and reliability. Nevertheless, their applicability for point-of-care testing (POCT) is limited due to the high cost of equipment, prolonged detection durations, and reliance on controlled laboratory settings. The CRISPR/Cas system is considered as the next-generation of nucleic acid-based molecular diagnostic techniques, attributed to its highly specific recognition of target nucleic acids, programmability, high sensitivity, and suitability for POCT. The one-pot CRISPR assay integrates the pre-amplification of DNA or RNA with CRISPR/Cas detection into a single reaction system. This consolidation streamlines experimental workflows, enhances the practicality of POCT, and minimizes the risk of aerosol contamination. Consequently, this method has received tremendous attention from researchers. However, challenges remain due to incompatibilities between isothermal amplification and the CRISPR/Cas system, including mutual interference, temperature mismatches, and buffer incompatibility. This paper reviews recent advancements in one-pot CRISPR/Cas detection strategies, highlighting potential solutions such as physical isolation, microfluidic integration, optimization of the reaction systems, modification of crRNA and PAM sequences and engineering of nucleases. Additionally, it explores the potential applications and challenges associated with these systems in the development of high-performance POCT platforms.