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

作者：Lin Mei, Zhiqiang Qiu, Mengen Hao, Weiwei Qi, Ting Zhang, Yuting Shen, Hongrui Xiao, Chaoyue Liang, Long-Xu Xie, Yongzhong Jiang, Meng Cheng, Tian Tian, Xiaoming Zhou · 发表于：Nature Communications · 年份：2025 · DOI：10.1038/s41467-025-60917-9 · 被引用次数：52 · 研究领域：CRISPR and Genetic Engineering、Advanced biosensing and bioanalysis techniques、Biosensors and Analytical Detection

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. Significant progr...