One-pot RPA/CRISPR-Cas12a assay with photomodulated aptamer-based inhibitors

Abstract

CRISPR diagnostic methods are attracting attention in nucleic acid testing (NAT) due to their sensitivity, specificity, and simplicity. To enhance the sensitivity and eliminate the risk of contamination associated with lid opening, a one-pot reaction combining the CRISPR system with recombinase polymerase amplification (RPA), a compatible amplification method, is used. However, the cleavage activity of the CRISPR system affects the efficiency of RPA, thereby limiting its effectiveness. In this study, we propose a one-pot RPA/CRISPR-Cas12a system that achieves high sensitivity through photomodulation. By utilizing a Cas12a inhibitory aptamer with a photocleavable (PC) linker, the Cas12a ribonucleoprotein can be temporarily inactivated, preventing it from degrading the RPA component during nucleic acid amplification. Upon completion of the amplification, brief UV irradiation can cleave the PC linker, allowing the Cas12a reaction to proceed and generate a fluorescence signal. This approach effectively eliminates the risk of contamination, achieves high specificity, and mitigates factors contributing to a reduction in sensitivity. Using this photomodulated one-pot RPA/CRISPR-Cas12a assay, we successfully detected the p72 gene of African swine fever virus at a concentration of 50 copies (2.5 copies/µL). This efficient one-pot assay exhibits high sensitivity, cost-effectiveness, simplicity, and a stable design, making it a highly promising technique for field diagnostics in NAT applications.