Multiplexed CRISPR Assay for Amplification-Free Detection of miRNAs
CRISPR-Cas proteins from bacteria are powerful tools for gene editing and molecular diagnostics. Expanding capacity of CRISPR to low cost, multiplexed assays of biomarkers is a key to future disease diagnostics, since multiple biomarker detection is essential for reliable diagnostics. Herein we desc...
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MDPI AG
2025-05-01
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| Series: | Biosensors |
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| Online Access: | https://www.mdpi.com/2079-6374/15/6/346 |
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| author | P. I. Thilini De Silva Keshani Hiniduma Rachelle Canete Ketki S. Bhalerao Sherif M. Shawky Hansana Gunathilaka Jessica L. Rouge Islam M. Mosa David C. Steffens Kevin Manning Breno S. Diniz James F. Rusling |
| author_facet | P. I. Thilini De Silva Keshani Hiniduma Rachelle Canete Ketki S. Bhalerao Sherif M. Shawky Hansana Gunathilaka Jessica L. Rouge Islam M. Mosa David C. Steffens Kevin Manning Breno S. Diniz James F. Rusling |
| author_sort | P. I. Thilini De Silva |
| collection | DOAJ |
| description | CRISPR-Cas proteins from bacteria are powerful tools for gene editing and molecular diagnostics. Expanding capacity of CRISPR to low cost, multiplexed assays of biomarkers is a key to future disease diagnostics, since multiple biomarker detection is essential for reliable diagnostics. Herein we describe a multiplexed assay in a 3D-printed 96-well plate with CRISPR-Cas13a immobilized in each well to target three circulating blood biomarker microRNAs (miRNAs 34c-5p, 200c-3p, and 30e-5p) for Alzheimer’s disease (ALZ). Immobilized Cas13a is equipped with different crRNAs complementary to each miRNA target. MiRNA binding to crRNA complements activates the collateral RNase activity of Cas13a, cleaving a quenched fluorescent reporter (RNaseAlert) with fluorophore and quencher connected by an RNA oligonucleotide to enable fluorescence measurements. We achieved ultralow limits of detection (LOD) of 0.74 fg/mL for miRNA 34c-5p, 0.70 fg/mL for miRNA 30e-5p, and 7.4 fg/mL for miRNA 200c-3p, with dynamic ranges from LODs up to about 1800 pg/mL. The accuracy of the assay was validated by spike-recovery studies and good correlation of levels of patient plasma samples vs. a referee method. This new approach provides selective, sensitive multiplex miRNA biosensing, and simultaneously accommodates analysis of standards and controls. |
| format | Article |
| id | doaj-art-9bad719b6b48452a9c4e3a4ef0727d49 |
| institution | Kabale University |
| issn | 2079-6374 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biosensors |
| spelling | doaj-art-9bad719b6b48452a9c4e3a4ef0727d492025-08-20T03:27:21ZengMDPI AGBiosensors2079-63742025-05-0115634610.3390/bios15060346Multiplexed CRISPR Assay for Amplification-Free Detection of miRNAsP. I. Thilini De Silva0Keshani Hiniduma1Rachelle Canete2Ketki S. Bhalerao3Sherif M. Shawky4Hansana Gunathilaka5Jessica L. Rouge6Islam M. Mosa7David C. Steffens8Kevin Manning9Breno S. Diniz10James F. Rusling11Department of Chemistry, University of Connecticut, Storrs, CT 06269, USADepartment of Chemistry, University of Connecticut, Storrs, CT 06269, USADepartment of Chemistry, University of Connecticut, Storrs, CT 06269, USADepartment of Chemistry, University of Connecticut, Storrs, CT 06269, USADepartment of Chemistry, University of Connecticut, Storrs, CT 06269, USADepartment of Chemistry, University of Connecticut, Storrs, CT 06269, USADepartment of Chemistry, University of Connecticut, Storrs, CT 06269, USADepartment of Chemistry, University of Connecticut, Storrs, CT 06269, USADepartment of Psychiatry, UConn Health, Farmington, CT 06030, USADepartment of Psychiatry, UConn Health, Farmington, CT 06030, USADepartment of Psychiatry, UConn Health, Farmington, CT 06030, USADepartment of Chemistry, University of Connecticut, Storrs, CT 06269, USACRISPR-Cas proteins from bacteria are powerful tools for gene editing and molecular diagnostics. Expanding capacity of CRISPR to low cost, multiplexed assays of biomarkers is a key to future disease diagnostics, since multiple biomarker detection is essential for reliable diagnostics. Herein we describe a multiplexed assay in a 3D-printed 96-well plate with CRISPR-Cas13a immobilized in each well to target three circulating blood biomarker microRNAs (miRNAs 34c-5p, 200c-3p, and 30e-5p) for Alzheimer’s disease (ALZ). Immobilized Cas13a is equipped with different crRNAs complementary to each miRNA target. MiRNA binding to crRNA complements activates the collateral RNase activity of Cas13a, cleaving a quenched fluorescent reporter (RNaseAlert) with fluorophore and quencher connected by an RNA oligonucleotide to enable fluorescence measurements. We achieved ultralow limits of detection (LOD) of 0.74 fg/mL for miRNA 34c-5p, 0.70 fg/mL for miRNA 30e-5p, and 7.4 fg/mL for miRNA 200c-3p, with dynamic ranges from LODs up to about 1800 pg/mL. The accuracy of the assay was validated by spike-recovery studies and good correlation of levels of patient plasma samples vs. a referee method. This new approach provides selective, sensitive multiplex miRNA biosensing, and simultaneously accommodates analysis of standards and controls.https://www.mdpi.com/2079-6374/15/6/346CRISPRAlzheimer biomarkersmiRNAsmultiplexed assayfluorescence |
| spellingShingle | P. I. Thilini De Silva Keshani Hiniduma Rachelle Canete Ketki S. Bhalerao Sherif M. Shawky Hansana Gunathilaka Jessica L. Rouge Islam M. Mosa David C. Steffens Kevin Manning Breno S. Diniz James F. Rusling Multiplexed CRISPR Assay for Amplification-Free Detection of miRNAs Biosensors CRISPR Alzheimer biomarkers miRNAs multiplexed assay fluorescence |
| title | Multiplexed CRISPR Assay for Amplification-Free Detection of miRNAs |
| title_full | Multiplexed CRISPR Assay for Amplification-Free Detection of miRNAs |
| title_fullStr | Multiplexed CRISPR Assay for Amplification-Free Detection of miRNAs |
| title_full_unstemmed | Multiplexed CRISPR Assay for Amplification-Free Detection of miRNAs |
| title_short | Multiplexed CRISPR Assay for Amplification-Free Detection of miRNAs |
| title_sort | multiplexed crispr assay for amplification free detection of mirnas |
| topic | CRISPR Alzheimer biomarkers miRNAs multiplexed assay fluorescence |
| url | https://www.mdpi.com/2079-6374/15/6/346 |
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