Competition and cooperation: A new idea for one-pot detection by isothermal amplification coupled with CRISPR

Rapid, highly sensitive, and specific nucleic acid detection technique has become a major focus in the field of biomedical detection. Isothermal nucleic acid amplification techniques (INAATs) offer rapid and sensitive nucleic acid amplification without the need for complex thermal cycling. Meanwhile, CRISPR-based systems provide a programmable and highly specific mechanism for nucleic acid recognition, utilizing the trans-cleavage activity of Cas proteins to generate a detectable signal. Combining the advantages of INAATs and CRISPR to achieve both high sensitivity and high specificity has therefore become a core research direction. One-pot detection can further reduce operational complexity and avoid aerosol contamination caused by repeatedly opening the lid. However, such integrated approaches still face several challenges, including false negatives due to template degradation, signal delays caused by cleavage of intermediate amplicons, and pervasive primer competition. In recent years, researchers have recognized these issues and developed various strategies to address them, such as spatial isolation, time-lapse separation, weaking CRISPR system activity, and system optimization. This review we systematically and comprehensively elucidate, for the first time, the competitive interactions inherent in isothermal amplification and CRISPR-based one-pot methods from a molecular mechanism perspective. Meanwhile, we discusses current strategies for enhancing the performance of one-pot assays, with the aim of supporting the development of novel technologies and facilitating the advancement of diagnostic reagents.