Cleavage Activity Adjustment of the CRISPR/Cas System for Enhanced Diagnostics

The CRISPR/Cas system, known for its high precision and efficiency in genome editing, has garnered significant interest and demonstrated potential in diagnostics, owing to advantages such as sensitivity and cost-effectiveness. However, the application of the CRISPR/Cas system in diagnostics is constrained by two inherent challenges. On one hand, the sensitivity of the CRISPR/Cas system for the direct detection of low-abundance targets remains to be further improved for the demands of practical detection. On the other hand, the integration of nucleic acid amplification with the CRISPR/Cas system in one-pot assays presents difficulty of balance between target amplification and cleavage of Cas proteins. To overcome these constraints, researchers have encouragingly developed diverse strategies for precisely adjusting the cleavage activity of the CRISPR/Cas system. This review systematically summarizes advancements across six major strategies, including protein engineering, crRNA engineering, activator engineering, reporter engineering, the influence of metal ions and small molecules, and aggregation-mediated enhancement. These approaches either enhance cleavage efficiency for amplification-free, ultrasensitive detection, or temporarily suppress the cleavage activity to coordinate kinetics in integrated one-pot systems. Furthermore, future perspectives are discussed, which will contribute to the development of cleavage activity adjustment of the CRISPR/Cas system, providing insights for next-generation diagnostic technologies.