Photocontrolled crRNA activation enables robust CRISPR-Cas12a diagnostics.

CRISPR diagnostics based on nucleic acid amplification faces barriers to its commercial use, such as contamination risks and insufficient sensitivity. Here, we propose a robust solution involving optochemical control of CRISPR RNA (crRNA) activation in CRISPR detection. Based on this strategy, recombinase polymerase amplification (RPA) and CRISPR-Cas12a detection systems can be integrated into a completely closed test tube. crRNA can be designed to be temporarily inactivated so that RPA is not affected by Cas12a cleavage. After the RPA reaction is completed, the CRISPR-Cas12a detection system is activated under rapid light irradiation. This photocontrolled, fully closed CRISPR diagnostic system avoids contamination risks and exhibits a more than two orders of magnitude improvement in sensitivity compared with the conventional one-pot assay. This photocontrolled CRISPR method was applied to the clinical detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, achieving detection sensitivity and specificity comparable to those of PCR. Furthermore, a compact and automatic photocontrolled CRISPR detection device was constructed.

Light-Driven Activation of RNA-Guided Nucleic Acid Cleavage.

As one of the most favorable stimuli, photoactivation provides an advantageous way to manipulate biological objects. In the current study, we have successfully demonstrated the use of light activation guide RNA (gRNA) strategy for controlling CRISPR systems. By conjugating photolabile protecting groups, the CRISPR functions became minimal, but exposure of acylated gRNAs to 365 nm light triggers the removal of masking groups, leading to the rescue of CRISPR functions. Furthermore, our strategy has been successfully used to control gene editing in human cells. This proof-of-concept study therefore demonstrates the promising potential of our strategy to versatile applications in chemical biology.