CrRNA Conformation-Engineered CRISPR-Cas12a System for Robust and Ultrasensitive Nucleic Acid Detection

Despite the widespread application of the CRISPR-Cas12a system in vitro diagnostics due to its high programmability and distinctive trans-cleavage activity, the susceptibility of its crRNA component to degradation and sensitivity to storage and working conditions poses a significant challenge to improving the practical efficacy of these diagnostic systems. Here, we show that engineered crRNA with a covalently closed circular structure (C-crRNA) can replace traditional linear crRNA to form functional complexes with Cas12a protein, significantly enhancing the anti-interference ability of the CRISPR-Cas12a system while maintaining its sensitivity and specificity. Based on this finding, a circular crRNA-mediated CRISPR molecular diagnostic (CRCD) toolkit is developed and successfully integrated with a standard nucleic acid amplification technique to detect synthesized Human Papillomavirus type 16 (HPV-16) plasmids down to 10 aM sensitivity levels. Furthermore, the CRCD system is applied for ultrasensitive detection of 40 HPV-16 and 40 influenza A viruses in clinical samples, with results consistent with those from PANTHER detection and quantitative real-time polymerase chain reaction (qRT-PCR). In conclusion, this strategy introduces a novel paradigm for engineering crRNA to program Cas12a, which has the potential to revolutionize the use of crRNA in CRISPR-based molecular diagnostics.