Dual-target CRISPR-Cas12 diagnostics based on asymmetrically chemical-modified DNA probe

CRISPR-based nucleic acid detection technologies have revolutionized infectious disease detection and environmental monitoring by leveraging RNA-DNA complementarity to enable rapid, precise, and cost-effective detection of targets. However, achieving multitarget detection in one tube still presents challenges that necessitate further research. Here, we develop a nucleic acid detection module based on the CRISPR-Cas12i system. Importantly, we find that Cas12i and AapCas12b exhibit opposite trans-cleavage preferences for asymmetrically phosphorothioate-modified single-strand DNA probes, enabling the development of an effective dual-target nucleic acid detection platform by combining these two Cas12 nucleases in one tube. Moreover, this dual-target detection platform exhibits high specificity and sensitivity in genotyping the nucleic acid targets of human papillomavirus (HPV) 16 and HPV18, as well as Influenza A virus (FluA) and Respiratory syncytial virus. Notably, combined with loop-mediated isothermal amplification, this platform achieves high detection rates for clinical samples (18/18 FluA and 18/18 GAPDH internal reference detection rate). Taken together, these results can broaden the application of CRISPR-based Cas12 proteins for multi-target nucleic acid detection in one tube.