Multiplexed CRISPR Assay for Amplification-Free Detection of miRNAs

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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Bibliographic Details
Main Authors: 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
Format: Article
Language:English
Published: MDPI AG 2025-05-01
Series:Biosensors
Subjects:
CRISPR
Alzheimer biomarkers
miRNAs
multiplexed assay
fluorescence
Online Access:https://www.mdpi.com/2079-6374/15/6/346
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Summary: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.
ISSN:2079-6374

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