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, Ye Hu
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. CRISPR-based diagnostics are often limited by complex workflows and poor validation. Here the authors develop and validate a one-pot asymmetric CRISPR assay, which permits rapid and sensitive diagnosis of tuberculosis in a format which is suitable for resource-limited settings.