Cas12a Cis-cleavage mediated lateral flow assay enables multiplex and ultra-specific nucleic acid detection

Mei Lin, Zhiqiang Qiu, Mengen Hao, Weiwei Qi, Ting Zhang, Yuting Shen, Hongrui Xiao, Chaoyue Liang, Longxu Xie, Yongzhong Jiang, Meng Cheng (), Tian Tian () and Xiaoming Zhou ()
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Mei Lin: South China Normal University
Zhiqiang Qiu: South China Normal University
Mengen Hao: South China Normal University
Weiwei Qi: South China Normal University
Ting Zhang: South China Normal University
Yuting Shen: South China Normal University
Hongrui Xiao: South China Normal University
Chaoyue Liang: South China Normal University
Longxu Xie: Guangzhou Hybribio Medicine Technology Ltd
Yongzhong Jiang: Hubei Provincial Center for Disease Control and Prevention
Meng Cheng: The First Affiliated Hospital of Guangzhou Medical University
Tian Tian: South China Normal University
Xiaoming Zhou: South China Normal University

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

Abstract: Abstract CRISPR technology holds significant promise for advancing nucleic acid assays. However, current CRISPR diagnostic techniques, reliant on indiscriminate trans-cleavage mechanisms, face challenges in developing multiplex detection formats. Moreover, chaotic trans-cleavage activity often results from mismatched targets, leading to specificity issues. To address these limitations, here we exploit a double-key recognition mechanism based on CRISPR-Cas12a cis-cleavage and invasive hybridization identification of released sticky-end DNA products. By integrating multiplexed nucleic acid amplification, the double-key Cas12a detection mechanism, and a lateral flow detection platform, we develop a method termed Cas12a cis-cleavage mediated lateral flow assay (cc-LFA). We demonstrate that the cc-LFA exhibited superior specificity compared to three mainstream trans-cleavage-based CRISPR diagnostic techniques, achieving single-base resolution detection free from high-concentration wild-type DNA background interference. cc-LFA is also applied for highly specific detection of multiple respiratory pathogen samples and precise multiplexed detection of nine high-risk human papillomavirus (HPV) subtypes, achieving over 90% sensitivity and 100% specificity, respectively. Additionally, we present a portable device to automate nucleic acid amplification and strip detection procedures, showcasing the potential of cc-LFA for future applications in decentralized laboratory scenarios.

Date: 2025
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DOI: 10.1038/s41467-025-60917-9

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