Citation: ZHANG Han-Xiao, DAI Xiao-Chun, ZHOU Ya-Nan, LYU Xu-Zhen, GONG Tao, ZHAO Xu-Hua, YU Bao-Feng. CRISPR/Cas12a Fluorescence Sensor Based on Molecular Beacon for Amplification Detection of Circular Tumor DNA[J]. Chinese Journal of Analytical Chemistry, ;2023, 51(2): 184-193. doi: 10.19756/j.issn.0253-3820.221203 shu

CRISPR/Cas12a Fluorescence Sensor Based on Molecular Beacon for Amplification Detection of Circular Tumor DNA

Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan 030001, China

Corresponding author: ZHAO Xu-Hua, YU Bao-Feng,
Received Date: 26 April 2022
Revised Date: 1 December 2022

Fund Project: Supported by the Natural Science Foundation of Shanxi Province, China (Nos. 202103021224240, 201901D211317, 201901D111190), the Open Fund from Key Laboratory of Cellular Physiology (Shanxi Medical University), Ministry of Education, China (No. KLMEC/SXMU-202011) and the Shanxi “1331 Project” Key Subjects Construction (No. 1331KSC).

A CRISPR/Cas12a-based biosensor using molecular beacon (MB) as the reporter was constructed for amplification detection of circular tumor DNA (ctDNA). The molecular beacon with good stability was labeled with FAM and TAMRA at its ends, respectively. In the absence of ctDNA, the CRISPR/Cas12a system was inactive and MB could not be cleaved. Therefore, two fluorophores were in close proximity to each other, resulting in fluorescence resonance energy transfer (FRET). In the presence of ctDNA, it could recognize the Cas12a/crRNA complex and activate the trans-cleavage activity of Cas12a. Because ssDNA was the most susceptible substrate of Cas12a, the loop of MB was rapidly cleaved first. After cleavage, two fluorophores were far from each other, leading to the disappearance of FRET phenomenon and an obvious fluorescent enhancement of FAM. The experimental conditions such as the base numbers of the hairpin loop, the concentration of MB, and the concentration ratio of crRNA to Cas12a were optimized. Under the optimal conditions, a linear relationship ranging from 1.7 pmol/L to 500 pmol/L for ctDNA quantitative detection was observed and its detection limit was 600 fmol/L. In addition, this sensor could be used to detect ctDNA in serum samples and the recoveries were in the range of 93%-110%.
- CRISPR,
- Biosensor,
- Fluorescence resonance energy transfer,
- Circular tumor DNA
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