Cas12a Cis-cleavage mediated lateral flow assay enables multiplex and ultra-specific nucleic acid detection
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 resul...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-07-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-60917-9 |
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| _version_ | 1849238445910654976 |
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| author | Mei Lin Zhiqiang Qiu Mengen Hao Weiwei Qi Ting Zhang Yuting Shen Hongrui Xiao Chaoyue Liang Longxu Xie Yongzhong Jiang Meng Cheng Tian Tian Xiaoming Zhou |
| author_facet | Mei Lin Zhiqiang Qiu Mengen Hao Weiwei Qi Ting Zhang Yuting Shen Hongrui Xiao Chaoyue Liang Longxu Xie Yongzhong Jiang Meng Cheng Tian Tian Xiaoming Zhou |
| author_sort | Mei Lin |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-25c176db76f64d27b3dec0ccbe619db2 |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-25c176db76f64d27b3dec0ccbe619db22025-08-20T04:01:36ZengNature PortfolioNature Communications2041-17232025-07-0116111410.1038/s41467-025-60917-9Cas12a Cis-cleavage mediated lateral flow assay enables multiplex and ultra-specific nucleic acid detectionMei Lin0Zhiqiang Qiu1Mengen Hao2Weiwei Qi3Ting Zhang4Yuting Shen5Hongrui Xiao6Chaoyue Liang7Longxu Xie8Yongzhong Jiang9Meng Cheng10Tian Tian11Xiaoming Zhou12School of Life sciences, South China Normal UniversitySchool of Life sciences, South China Normal UniversitySchool of Life sciences, South China Normal UniversitySchool of Life sciences, South China Normal UniversitySchool of Life sciences, South China Normal UniversitySchool of Life sciences, South China Normal UniversitySchool of Life sciences, South China Normal UniversitySchool of Life sciences, South China Normal UniversityGuangzhou Hybribio Medicine Technology LtdHubei Provincial Center for Disease Control and PreventionDepartment of Clinical Laboratory, The First Affiliated Hospital of Guangzhou Medical UniversitySchool of Life sciences, South China Normal UniversitySchool of Life sciences, South China Normal UniversityAbstract 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.https://doi.org/10.1038/s41467-025-60917-9 |
| spellingShingle | Mei Lin Zhiqiang Qiu Mengen Hao Weiwei Qi Ting Zhang Yuting Shen Hongrui Xiao Chaoyue Liang Longxu Xie Yongzhong Jiang Meng Cheng Tian Tian Xiaoming Zhou Cas12a Cis-cleavage mediated lateral flow assay enables multiplex and ultra-specific nucleic acid detection Nature Communications |
| title | Cas12a Cis-cleavage mediated lateral flow assay enables multiplex and ultra-specific nucleic acid detection |
| title_full | Cas12a Cis-cleavage mediated lateral flow assay enables multiplex and ultra-specific nucleic acid detection |
| title_fullStr | Cas12a Cis-cleavage mediated lateral flow assay enables multiplex and ultra-specific nucleic acid detection |
| title_full_unstemmed | Cas12a Cis-cleavage mediated lateral flow assay enables multiplex and ultra-specific nucleic acid detection |
| title_short | Cas12a Cis-cleavage mediated lateral flow assay enables multiplex and ultra-specific nucleic acid detection |
| title_sort | cas12a cis cleavage mediated lateral flow assay enables multiplex and ultra specific nucleic acid detection |
| url | https://doi.org/10.1038/s41467-025-60917-9 |
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