Multiplex gRNAs Synergically Enhance Detection of SARS-CoV-2 by CRISPR-Cas12a.
CRISPR J
; 6(2): 116-126, 2023 04.
Article
in English
| MEDLINE | ID: covidwho-2272837
ABSTRACT
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) diagnostic methods have a large potential to effectively detect SARS-CoV-2 with sensitivity and specificity nearing 100%, comparable to quantitative polymerase chain reaction. Yet, there is room for improvement. Commonly, one guide CRISPR RNA (gRNA) is used to detect the virus DNA and activate Cas collateral activity, which cleaves a reporter probe. In this study, we demonstrated that using 2-3 gRNAs in parallel can create a synergistic effect, resulting in a 4.5 × faster cleaving rate of the probe and increased sensitivity compared to using individual gRNAs. The synergy is due to the simultaneous activation of CRISPR-Cas12a and the improved performance of each gRNA. This approach was able to detect as few as 10 viral copies of the N-gene of SARS-CoV-2 RNA after a preamplification step using reverse transcription loop-mediated isothermal amplification. The method was able to accurately detect 100% of positive and negative clinical samples in â¼25 min using a fluorescence plate reader and â¼45 min with lateral flow strips.
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
SARS-CoV-2
/
COVID-19
Type of study:
Diagnostic study
/
Prognostic study
Limits:
Humans
Language:
English
Journal:
CRISPR J
Year:
2023
Document Type:
Article
Affiliation country:
Crispr.2022.0074
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