RNase H‑Driven crRNA Switch Circuits for Rapid and Sensitive Detection of Various Analytical Targets

The clustered regularly interspaced short palindromic repeats (CRISPR/Cas12a) system has exhibited great promise in the rapid and sensitive molecular diagnostics for its trans-cleavage property. However, most CRISPR/Cas system-based detection methods are designed for nucleic acids and require target...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2023-12, Vol.95 (50), p.18549-18556
Main Authors: Li, Hua-Dong, Fang, Guan-Hua, Ye, Bang-Ce, Yin, Bin-Cheng
Format: Article
Language:English
Subjects:
Adenosine triphosphate
ATP
Biosensors
Circuits
Cleavage
CRISPR
Deoxyribonucleic acid
Detection limits
DNA
Nanotechnology
Nucleic acids
p53 Protein
Ribonuclease H
Target detection
Thrombin
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Summary:The clustered regularly interspaced short palindromic repeats (CRISPR/Cas12a) system has exhibited great promise in the rapid and sensitive molecular diagnostics for its trans-cleavage property. However, most CRISPR/Cas system-based detection methods are designed for nucleic acids and require target preamplification to improve sensitivity and detection limits. Here, we propose a generic crRNA switch circuit-regulated CRISPR/Cas sensor for the sensitive detection of various targets. The crRNA switch is engineered and designed in a blocked state but can be activated in the presence of triggers, which are target-induced association DNA to initiate the trans-cleavage activity of Cas12a for signal reporting. Additionally, RNase H is introduced to specifically hydrolyze RNA duplexed with the DNA trigger, resulting in the regeneration of the trigger to activate more crRNA switches. Such a combination provides a generic and sensitive strategy for the effective sensing of the p53 sequence, thrombin, and adenosine triphosphate. The design is incorporated with nucleic acid nanotechnology and extensively broadens the application scope of the CRISPR technology in biosensing.
ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.3c04267