A protospacer adjacent motif‐free, multiplexed, and quantitative nucleic acid detection platform with barcode‐based Cas12a activity
Abstract Clustered regularly interspaced short palindromic repeat (CRISPR)‐based biosensors have been developed to facilitate the rapid and sensitive detection of nucleic acids. However, most approaches using CRISPR‐based detection have disadvantages associated with the limitations of CRISPR RNA (cr...
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| Format: | Article |
| Language: | English |
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Wiley
2023-08-01
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| Series: | MedComm |
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| Online Access: | https://doi.org/10.1002/mco2.310 |
| _version_ | 1797693049603620864 |
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| author | Miaojin Zhou Chunhua Zhang Miaomiao Chen Zhiqing Hu Menglin Li Zhuo Li Lingqian Wu Desheng Liang |
| author_facet | Miaojin Zhou Chunhua Zhang Miaomiao Chen Zhiqing Hu Menglin Li Zhuo Li Lingqian Wu Desheng Liang |
| author_sort | Miaojin Zhou |
| collection | DOAJ |
| description | Abstract Clustered regularly interspaced short palindromic repeat (CRISPR)‐based biosensors have been developed to facilitate the rapid and sensitive detection of nucleic acids. However, most approaches using CRISPR‐based detection have disadvantages associated with the limitations of CRISPR RNA (crRNA), protospacer adjacent motif (PAM) or protospacer flanking sequence restriction, single channel detection, and difficulty in quantitative detection resulting in only some target sites being detected qualitatively. Here, we aimed to develop a barcode‐based Cas12a‐mediated DNA detection (BCDetection) strategy, which overcomes the aforementioned drawbacks and enables (1) detection with a universal PAM and crRNA without PAM or crRNA restriction, (2) simultaneous detection of multiple targets in a single reaction, and (3) quantitative detection, which can significantly distinguish copy number differences up to as low as a two‐fold limit. We could efficiently and simultaneously detect three β‐thalassemia mutations in a single reaction using BCDetection. Notably, samples from normal individuals, spinal muscular atrophy (SMA) carriers, and SMA patients were significantly and accurately distinguished using the quantitative detection ability of BCDetection, indicating its potential application in β‐thalassemia and SMA carrier screening. Therefore, our findings demonstrate that BCDetection provides a new platform for accurate and efficient quantitative detection using CRISPR/Cas12a, highlighting its bioanalytical applications. |
| first_indexed | 2024-03-12T02:37:57Z |
| format | Article |
| id | doaj.art-087db16c417e4d0db4f6b52e1ff8360f |
| institution | Directory Open Access Journal |
| issn | 2688-2663 |
| language | English |
| last_indexed | 2024-03-12T02:37:57Z |
| publishDate | 2023-08-01 |
| publisher | Wiley |
| record_format | Article |
| series | MedComm |
| spelling | doaj.art-087db16c417e4d0db4f6b52e1ff8360f2023-09-04T11:20:36ZengWileyMedComm2688-26632023-08-0144n/an/a10.1002/mco2.310A protospacer adjacent motif‐free, multiplexed, and quantitative nucleic acid detection platform with barcode‐based Cas12a activityMiaojin Zhou0Chunhua Zhang1Miaomiao Chen2Zhiqing Hu3Menglin Li4Zhuo Li5Lingqian Wu6Desheng Liang7Center for Medical Genetics & Hunan Key Laboratory of Medical Genetics School of Life Sciences Central South University Changsha Hunan ChinaCenter for Medical Genetics & Hunan Key Laboratory of Medical Genetics School of Life Sciences Central South University Changsha Hunan ChinaCenter for Medical Genetics & Hunan Key Laboratory of Medical Genetics School of Life Sciences Central South University Changsha Hunan ChinaCenter for Medical Genetics & Hunan Key Laboratory of Medical Genetics School of Life Sciences Central South University Changsha Hunan ChinaCenter for Medical Genetics & Hunan Key Laboratory of Medical Genetics School of Life Sciences Central South University Changsha Hunan ChinaCenter for Medical Genetics & Hunan Key Laboratory of Medical Genetics School of Life Sciences Central South University Changsha Hunan ChinaCenter for Medical Genetics & Hunan Key Laboratory of Medical Genetics School of Life Sciences Central South University Changsha Hunan ChinaCenter for Medical Genetics & Hunan Key Laboratory of Medical Genetics School of Life Sciences Central South University Changsha Hunan ChinaAbstract Clustered regularly interspaced short palindromic repeat (CRISPR)‐based biosensors have been developed to facilitate the rapid and sensitive detection of nucleic acids. However, most approaches using CRISPR‐based detection have disadvantages associated with the limitations of CRISPR RNA (crRNA), protospacer adjacent motif (PAM) or protospacer flanking sequence restriction, single channel detection, and difficulty in quantitative detection resulting in only some target sites being detected qualitatively. Here, we aimed to develop a barcode‐based Cas12a‐mediated DNA detection (BCDetection) strategy, which overcomes the aforementioned drawbacks and enables (1) detection with a universal PAM and crRNA without PAM or crRNA restriction, (2) simultaneous detection of multiple targets in a single reaction, and (3) quantitative detection, which can significantly distinguish copy number differences up to as low as a two‐fold limit. We could efficiently and simultaneously detect three β‐thalassemia mutations in a single reaction using BCDetection. Notably, samples from normal individuals, spinal muscular atrophy (SMA) carriers, and SMA patients were significantly and accurately distinguished using the quantitative detection ability of BCDetection, indicating its potential application in β‐thalassemia and SMA carrier screening. Therefore, our findings demonstrate that BCDetection provides a new platform for accurate and efficient quantitative detection using CRISPR/Cas12a, highlighting its bioanalytical applications.https://doi.org/10.1002/mco2.310barcode‐based Cas12acopy number variationsimultaneous detection of multiple targetsspinal muscular atrophyuniversal PAM and crRNA |
| spellingShingle | Miaojin Zhou Chunhua Zhang Miaomiao Chen Zhiqing Hu Menglin Li Zhuo Li Lingqian Wu Desheng Liang A protospacer adjacent motif‐free, multiplexed, and quantitative nucleic acid detection platform with barcode‐based Cas12a activity MedComm barcode‐based Cas12a copy number variation simultaneous detection of multiple targets spinal muscular atrophy universal PAM and crRNA |
| title | A protospacer adjacent motif‐free, multiplexed, and quantitative nucleic acid detection platform with barcode‐based Cas12a activity |
| title_full | A protospacer adjacent motif‐free, multiplexed, and quantitative nucleic acid detection platform with barcode‐based Cas12a activity |
| title_fullStr | A protospacer adjacent motif‐free, multiplexed, and quantitative nucleic acid detection platform with barcode‐based Cas12a activity |
| title_full_unstemmed | A protospacer adjacent motif‐free, multiplexed, and quantitative nucleic acid detection platform with barcode‐based Cas12a activity |
| title_short | A protospacer adjacent motif‐free, multiplexed, and quantitative nucleic acid detection platform with barcode‐based Cas12a activity |
| title_sort | protospacer adjacent motif free multiplexed and quantitative nucleic acid detection platform with barcode based cas12a activity |
| topic | barcode‐based Cas12a copy number variation simultaneous detection of multiple targets spinal muscular atrophy universal PAM and crRNA |
| url | https://doi.org/10.1002/mco2.310 |
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