CRISPR/Cas13a-assisted AMP generation for SARS-CoV-2 RNA detection using a personal glucose meter
Herein, we described a washing- and label-free clustered regularly interspaced short palindromic repeats (CRISPR)/LwaCas13a-based RNA detection method utilizing a personal glucose meter (PGM), which relies on the trans-cleavage activity of CRISPR/Cas13a and kinase reactions. In principle, the presen...
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| Format: | Article |
| Language: | English |
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Elsevier
2022-12-01
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| Series: | Biosensors and Bioelectronics: X |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2590137022001765 |
| _version_ | 1828165539392913408 |
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| author | Junhyun Park Hyogu Han Jae Hoon Jeung Hyowon Jang Chihyun Park Jun Ki Ahn |
| author_facet | Junhyun Park Hyogu Han Jae Hoon Jeung Hyowon Jang Chihyun Park Jun Ki Ahn |
| author_sort | Junhyun Park |
| collection | DOAJ |
| description | Herein, we described a washing- and label-free clustered regularly interspaced short palindromic repeats (CRISPR)/LwaCas13a-based RNA detection method utilizing a personal glucose meter (PGM), which relies on the trans-cleavage activity of CRISPR/Cas13a and kinase reactions. In principle, the presence of target RNA activates the trans-cleavage of CRISPR/Cas13a, generating 2′,3′-cyclic phosphate adenosine, which is converted to adenosine monophosphate (AMP) by the T4 polynucleotide kinase. Subsequently, the AMP is converted to adenosine diphosphate (ADP) through phosphorylation by a myokinase; ADP is then used as a substrate in the cascade enzymatic reaction promoted by pyruvate kinase and hexokinase. The overall reaction leads to the continuous conversion of glucose to glucose-6-phosphate, resulting in a reduction of glucose concentration proportional to the level of target RNA, which can therefore be indirectly measured with a PGM. By employing this novel strategy, severe acute respiratory syndrome coronavirus-2 RNA can be successfully detected with excellent specificity. In addition, we were able to overcome non-specific responses of CRISPR/Cas13a and distinguish single nucleotide polymorphisms by introducing a single-base mismatch in the complementary RNA. Our study provides an alternative coronavirus disease 2019 detection technology that is affordable, accessible, and portable with a fast turnaround time and excellent selectivity. |
| first_indexed | 2024-04-12T01:45:11Z |
| format | Article |
| id | doaj.art-b67ebdfb6a934b9caae9005f20ede984 |
| institution | Directory Open Access Journal |
| issn | 2590-1370 |
| language | English |
| last_indexed | 2024-04-12T01:45:11Z |
| publishDate | 2022-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Biosensors and Bioelectronics: X |
| spelling | doaj.art-b67ebdfb6a934b9caae9005f20ede9842022-12-22T03:53:05ZengElsevierBiosensors and Bioelectronics: X2590-13702022-12-0112100283CRISPR/Cas13a-assisted AMP generation for SARS-CoV-2 RNA detection using a personal glucose meterJunhyun Park0Hyogu Han1Jae Hoon Jeung2Hyowon Jang3Chihyun Park4Jun Ki Ahn5Material & Component Convergence R&D Department, Korea Institute of Industrial Technology (KITECH), Ansan, 15588, South Korea; Department of Biological Engineering, College of Engineering, Konkuk University, Seoul, 05029, South KoreaMaterial & Component Convergence R&D Department, Korea Institute of Industrial Technology (KITECH), Ansan, 15588, South Korea; Department of Chemistry, Gangneung-Wonju National University, Gangneung, 25457, South KoreaMaterial & Component Convergence R&D Department, Korea Institute of Industrial Technology (KITECH), Ansan, 15588, South Korea; Department of Biological Engineering, College of Engineering, Konkuk University, Seoul, 05029, South KoreaBionanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yuseong-gu, Daejeon, 34141, South KoreaDaejeon District Office, National Forensic Service, Daejeon, 34054, South KoreaMaterial & Component Convergence R&D Department, Korea Institute of Industrial Technology (KITECH), Ansan, 15588, South Korea; Corresponding author.Herein, we described a washing- and label-free clustered regularly interspaced short palindromic repeats (CRISPR)/LwaCas13a-based RNA detection method utilizing a personal glucose meter (PGM), which relies on the trans-cleavage activity of CRISPR/Cas13a and kinase reactions. In principle, the presence of target RNA activates the trans-cleavage of CRISPR/Cas13a, generating 2′,3′-cyclic phosphate adenosine, which is converted to adenosine monophosphate (AMP) by the T4 polynucleotide kinase. Subsequently, the AMP is converted to adenosine diphosphate (ADP) through phosphorylation by a myokinase; ADP is then used as a substrate in the cascade enzymatic reaction promoted by pyruvate kinase and hexokinase. The overall reaction leads to the continuous conversion of glucose to glucose-6-phosphate, resulting in a reduction of glucose concentration proportional to the level of target RNA, which can therefore be indirectly measured with a PGM. By employing this novel strategy, severe acute respiratory syndrome coronavirus-2 RNA can be successfully detected with excellent specificity. In addition, we were able to overcome non-specific responses of CRISPR/Cas13a and distinguish single nucleotide polymorphisms by introducing a single-base mismatch in the complementary RNA. Our study provides an alternative coronavirus disease 2019 detection technology that is affordable, accessible, and portable with a fast turnaround time and excellent selectivity.http://www.sciencedirect.com/science/article/pii/S2590137022001765SARS-CoV-2CRISPR/Cas13aKinaseCascade enzymatic reactionPersonal glucose meterBiosensor |
| spellingShingle | Junhyun Park Hyogu Han Jae Hoon Jeung Hyowon Jang Chihyun Park Jun Ki Ahn CRISPR/Cas13a-assisted AMP generation for SARS-CoV-2 RNA detection using a personal glucose meter Biosensors and Bioelectronics: X SARS-CoV-2 CRISPR/Cas13a Kinase Cascade enzymatic reaction Personal glucose meter Biosensor |
| title | CRISPR/Cas13a-assisted AMP generation for SARS-CoV-2 RNA detection using a personal glucose meter |
| title_full | CRISPR/Cas13a-assisted AMP generation for SARS-CoV-2 RNA detection using a personal glucose meter |
| title_fullStr | CRISPR/Cas13a-assisted AMP generation for SARS-CoV-2 RNA detection using a personal glucose meter |
| title_full_unstemmed | CRISPR/Cas13a-assisted AMP generation for SARS-CoV-2 RNA detection using a personal glucose meter |
| title_short | CRISPR/Cas13a-assisted AMP generation for SARS-CoV-2 RNA detection using a personal glucose meter |
| title_sort | crispr cas13a assisted amp generation for sars cov 2 rna detection using a personal glucose meter |
| topic | SARS-CoV-2 CRISPR/Cas13a Kinase Cascade enzymatic reaction Personal glucose meter Biosensor |
| url | http://www.sciencedirect.com/science/article/pii/S2590137022001765 |
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