1. One-base-mismatch CRISPR-based transistors for single nucleotide resolution assay.
- Author
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Ma H, Tian Y, Kong D, Guo M, Dai C, Wang Q, Li S, Tian Z, Liu Y, and Wei D
- Subjects
- Humans, SARS-CoV-2 genetics, SARS-CoV-2 isolation & purification, Base Pair Mismatch, Limit of Detection, COVID-19 virology, COVID-19 diagnosis, Graphite chemistry, Biosensing Techniques instrumentation, Biosensing Techniques methods, Transistors, Electronic, CRISPR-Cas Systems genetics, RNA, Viral genetics, RNA, Viral isolation & purification, RNA, Viral analysis, Polymorphism, Single Nucleotide genetics
- Abstract
An effective strategy for accurately detecting single nucleotide variants (SNVs) is of great significance for genetic research and diagnostics. However, strict amplification conditions, complex experimental instruments, and specialized personnel are required to obtain a satisfactory tradeoff between sensitivity and selectivity for SNV discrimination. In this study, we present a CRISPR-based transistor biosensor for the rapid and highly selective detection of SNVs in viral RNA. By introducing a synthetic mismatch in the crRNA, the CRISPR-Cas13a protein can be engineered to capture the target SNV RNA directly on the surface of the graphene channel. This process induces a fast electrical signal response in the transistor, obviating the need for amplification or reporter molecules. The biosensor exhibits a detection limit for target RNA as low as 5 copies in 100 μL, which is comparable to that of real-time quantitative polymerase chain reaction (PCR). Its operational range spans from 10 to 5 × 10
5 copy mL-1 in artificial saliva solution. This capability enables the biosensor to discriminate between wild-type and SNV RNA within 15 min. By introducing 10 μL of swab samples during clinical testing, the biosensor provides specific detection of respiratory viruses in 19 oropharyngeal specimens, including influenza A, influenza B, and variants of SARS-CoV-2. This study emphasizes the CRISPR-transistor technique as a highly accurate and sensitive approach for field-deployable nucleic acid screening or diagnostics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)- Published
- 2024
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