Sensitive pathogen DNA detection by a multi-guide RNA Cas12a assay favoring trans- versus cis-cleavage

Zhen Huang (), Zhe Song, Jianfeng Zeng, Xuhui Liu, Mutong Fang, Zhiyuan Wu, Yao Zhao, Yanli Chen, Dan Li, Huan Huang, Liang Fu, Peng Xu, Bo Ning, Jun Chen, Ming Guan, Lin Sun, Christopher J. Lyon, Xiao-Yong Fan, Shuihua Lu () and Tony Hu ()
Additional contact information
Zhen Huang: Tulane University School of Medicine
Zhe Song: Southern University of Science and Technology
Jianfeng Zeng: Southern University of Science and Technology
Xuhui Liu: Fudan University
Mutong Fang: Southern University of Science and Technology
Zhiyuan Wu: Fudan University
Yao Zhao: Southern University of Science and Technology
Yanli Chen: Fudan University
Dan Li: Southern University of Science and Technology
Huan Huang: Tulane University School of Medicine
Liang Fu: Southern University of Science and Technology
Peng Xu: Southern University of Science and Technology
Bo Ning: Tulane University School of Medicine
Jun Chen: Fudan University
Ming Guan: Fudan University
Lin Sun: Beijing Children’s Hospital, Capital Medical University, National Clinical Research Center for Respiratory Diseases, National Center for Children’s Health
Christopher J. Lyon: Tulane University School of Medicine
Xiao-Yong Fan: Fudan University
Shuihua Lu: Southern University of Science and Technology
Tony Hu: Tulane University School of Medicine

Nature Communications, 2025, vol. 16, issue 1, 1-14

Abstract: Abstract Most CRISPR assays lack clinical utility due to their complex workflows and limited validation. Here we present a streamlined “one-pot” asymmetric CRISPR tuberculosis assay that attenuates amplicon degradation to achieve 5 copies/μL sensitivity within 60 min and detect positive patient samples within 15 min. This assay exhibited 93%, 83%, and 93% sensitivity with adult respiratory, pediatric stool, and adult cerebral spinal fluid specimens, and detected 64% of clinically diagnosed tuberculous meningitis cases, in a cohort of 603 clinical samples. This assay achieves complete specificity and greater sensitivity (74% vs. 56%) than the most sensitive reference test with prospectively collected tongue swabs, and exhibits similar performance when adapted to a lateral flow assay format and employed to analyze self-collected tongue swabs. These results demonstrate the utility of this approach across diverse specimen types, including those suitable for use in remote and resource-limited settings, to improve access to molecular diagnostics.

Date: 2025
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DOI: 10.1038/s41467-025-63094-x

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