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An RNA-based catalytic hairpin assembly circuit coupled with CRISPR-Cas12a for one-step detection of microRNAs.

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Title: An RNA-based catalytic hairpin assembly circuit coupled with CRISPR-Cas12a for one-step detection of microRNAs.
Authors: Chen, Pinru1 (AUTHOR), Wang, Luying1 (AUTHOR), Qin, Peipei1 (AUTHOR), Yin, Bin-Cheng1,2,3 (AUTHOR) binchengyin@ecust.edu.cn, Ye, Bang-Ce1,2 (AUTHOR) bcye@ecust.edu.cn
Source: Biosensors & Bioelectronics. Jul2022, Vol. 207, pN.PAG-N.PAG. 1p.
Subjects: Hairpin (Genetics), MicroRNA, Detection limit, Molecular diagnosis, Cell lines, Nucleic acids, CRISPRs
Abstract: CRISPR-Cas nuclease-based nucleic acid detection has exhibited extraordinary value in the field of molecular diagnostics, but it usually involves two separate reaction steps of nucleic acid amplification and Cas-based endpoint detection, resulting in the use of multiple enzymes, inconvenient operation, and potential carry-over contamination. Here, we propose an RNA-based catalytic hairpin assembly (CHA) circuit coupled with CRISPR-Cas12a for one-step detection of microRNAs (miRNAs) at an isothermal condition. This method relies on the rational design of a spacer-blocking crRNA as a bridge between the two systems. The target miRNA can specifically trigger RNA-based CHA and induce a configurational change of the blocked crRNAs into precursor crRNAs (pre-crRNAs), which can be processed into mature crRNAs to function by leveraging the inherent RNase activities of Cas12a. In this way, the developed circuit achieves a femtomolar detection limit and shows an accurate detection of miRNA levels in different cell lines. Therefore, our method would provide a new paradigm to develop miRNA detection methods based on the CRISPR/Cas system. • A method for one-step microRNA detection based on CRISPR-Cas12a-assisted catalytic hairpin assembly (CHA) was developed. • The RNase and DNase activities of Cas12a were fully exploited. • It integrated the inherently high sensitivity and specificity both from CHA and CRISPR-Cas12a system. • This method was able to detect target miRNA down to femtomolar concentration. [ABSTRACT FROM AUTHOR]
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Database: Engineering Source
Description
Abstract:CRISPR-Cas nuclease-based nucleic acid detection has exhibited extraordinary value in the field of molecular diagnostics, but it usually involves two separate reaction steps of nucleic acid amplification and Cas-based endpoint detection, resulting in the use of multiple enzymes, inconvenient operation, and potential carry-over contamination. Here, we propose an RNA-based catalytic hairpin assembly (CHA) circuit coupled with CRISPR-Cas12a for one-step detection of microRNAs (miRNAs) at an isothermal condition. This method relies on the rational design of a spacer-blocking crRNA as a bridge between the two systems. The target miRNA can specifically trigger RNA-based CHA and induce a configurational change of the blocked crRNAs into precursor crRNAs (pre-crRNAs), which can be processed into mature crRNAs to function by leveraging the inherent RNase activities of Cas12a. In this way, the developed circuit achieves a femtomolar detection limit and shows an accurate detection of miRNA levels in different cell lines. Therefore, our method would provide a new paradigm to develop miRNA detection methods based on the CRISPR/Cas system. • A method for one-step microRNA detection based on CRISPR-Cas12a-assisted catalytic hairpin assembly (CHA) was developed. • The RNase and DNase activities of Cas12a were fully exploited. • It integrated the inherently high sensitivity and specificity both from CHA and CRISPR-Cas12a system. • This method was able to detect target miRNA down to femtomolar concentration. [ABSTRACT FROM AUTHOR]
ISSN:09565663
DOI:10.1016/j.bios.2022.114152