Conditionally Activated Cross-Linked crRNAs for CRISPR/Cas12a Based Nucleic Acid Detection

CRISPR/Cas systems, particularly CRISPR/Cas12a, have revolutionized nucleic acid detection due to their exceptional specificity and sensitivity. However, CRISPR/Cas12a's cleavage activity can interfere with amplification processes, such as reverse transcription (RT) and isothermal amplification (e.g., RPA), potentially compromising detection sensitivity and accuracy. While modified CRISPR/Cas12a systems employing caging and decaging strategies have been developed to address this, these approaches typically require extensive optimization of photolabile groups and complex assay configurations. Here, we present a universal, photochemically controlled strategy for CRISPR/Cas12a-based detection that overcomes these challenges. Our approach involves cross-linking a polymeric crRNA with a photoresponsive cross-linker, effectively inactivating it during amplification and enabling rapid activation through brief light exposure to cleave the cross-linker and release active crRNA. This method obviates the need for labor-intensive optimizations and modifications, making it highly versatile and suitable for rapid, on-site detection applications. Our strategy demonstrates enhanced versatility and applicability, particularly for the immediate detection of newly emerging or unexpected nucleic acid sequences, supporting applications in pathogen detection, genetic screening, and point-of-care diagnostics.