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Multiplexed CRISPR Assay for Amplification-Free Detection of miRNAs
De Silva PIT, Hiniduma K, Canete R, Bhalerao KS, Shawky SM, Gunathilaka H, Rouge JL, Mosa IM et al.. Biosensors, 2025-05-29
Topic: Brain proteins, microglia and lab models
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.
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APA 7th
De Silva, P. I. T., Hiniduma, K., Canete, R., Bhalerao, K. S., Shawky, S. M., Gunathilaka, H., et al. (2025). Multiplexed CRISPR Assay for Amplification-Free Detection of miRNAs. Biosensors. https://doi.org/10.3390/bios15060346
Vancouver
De Silva PIT, Hiniduma K, Canete R, Bhalerao KS, Shawky SM, Gunathilaka H, et al. Multiplexed CRISPR Assay for Amplification-Free Detection of miRNAs. Biosensors. 2025. doi:10.3390/bios15060346. PMID: 40558428.
BibTeX
@article{desilva2025-40558428,
author = {De Silva, P. I. T. and Hiniduma, K. and Canete, R. and Bhalerao, K. S. and Shawky, S. M. and Gunathilaka, H. and Rouge, J. L. and Mosa, I. M. and others},
title = {{Multiplexed CRISPR Assay for Amplification-Free Detection of miRNAs}},
journal = {Biosensors},
year = {2025},
doi = {10.3390/bios15060346},
pmid = {40558428},
url = {https://doi.org/10.3390/bios15060346}
}
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Key findings
- 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.
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Study details
- Evidence strengthLow
How this study scored 45/100
Published in a peer-reviewed journal. Scored zero for study design, sample size, effect reported, studied in people and randomised / blinded because the record does not state it. We have no data on journal's citation record, times cited, citations per year, impact for its field and year, cited by studies here, still being cited, senior author's track record, institution's track record and multi-centre collaboration, so those were left out: it earned 26 of the 58 points we could judge it on, which is 45 out of 100.
| Component | Points | Why |
|---|---|---|
| Journal tier | 7 / 10 | Tier 3 — peer-reviewed journal (Biosensors) |
| Journal's citation record | — / 4 | No journal record from OpenAlex — left out of this score |
| Study design | 0 / 12 | No study design recorded — scores zero, not guessed (not stated in the record — scored 0) |
| Strength of evidence | 1 / 5 | Rated “low” in our summary |
| Peer-review validation | 10 / 10 | Peer-reviewed. Published as an article in a named peer-reviewed journal. |
| How recent | 4 / 5 | 2025 — within 3 year(s) of the newest study here |
| Sample size | 0 / 4 | Participant count not stated — scores zero (not stated in the record — scored 0) |
| Effect reported | 0 / 3 | No effect size reported — scores zero (not stated in the record — scored 0) |
| Studied in people | 0 / 3 | Not stated whether this was studied in people — scores zero (not stated in the record — scored 0) |
| Randomised / blinded | 0 / 2 | No randomisation or blinding stated — scores zero (not stated in the record — scored 0) |
| Times cited | — / 7 | No citation count (not found in OpenAlex) — left out of this score |
| Citations per year | — / 5 | No citation count or no year — left out of this score |
| Impact for its field and year | — / 6 | No field-weighted citation impact (OpenAlex has none yet) — left out of this score |
| Cited by studies here | — / 6 | Not yet checked against this library's citation graph — left out of this score |
| Still being cited | — / 4 | No yearly citation counts — left out of this score |
| Senior author's track record | — / 4 | Senior author's record not available — left out of this score |
| Institution's track record | — / 3 | No institution listed in OpenAlex — left out of this score |
| Multi-centre collaboration | — / 3 | Institutions not listed — left out of this score |
| Indexed in PubMed | 2 / 2 | Indexed in PubMed (PMID 40558428) |
| Free to read | 2 / 2 | Free to read |
| Points | 26 / 58 | Scored on 11 of 20 parts. We have no data for the rest, so they are left out: 26 of the 58 points we could judge it on is 45 out of 100. |
| Total | 45 / 100 | — |
What each of these 20 components measures, and what this score is not →
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