a. State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China;
b. University of Chinese Academy of Sciences, Beijing 100049, China;
c. Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China;
d. SynsorBio Technology Co. Ltd Beijing, Beijing 100176, China;
e. Department of Clinical Laboratory, Shenshan Central Hospital, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Shanwei, Guangdong 510120, China;
f. Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Guangdong-Hong Kong Joint Laboratory for RNA Medicine, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, Guangdong 510120, China
Funds:
This work was supported by the Agriculture Science and Technology Major Project, the National Key Research and Development Program (2020YFA0707900 to X.W.
2022YFC2601200, 2023YFC2604904), the National Natural Science Foundation of China (32225030 and 82488301 to W.L.), the CAS Project for Young Scientists in Basic Research (YSBR-012 to W.L.), the China Postdoctoral Science Foundation (2023M743477 to Y.C.), and the Postdoctoral Fellowship Program of CPSF (GZB20230751 to Y.C.).
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.
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