國家衛生研究院 NHRI:Item 3990099045/16226
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    Please use this identifier to cite or link to this item: http://ir.nhri.org.tw/handle/3990099045/16226


    Title: Activated single nucleotide mismatch determination through toehold-embedded hairpin-mediated strand displacement reaction alongside CRISPR-Cas12a for detection of TP53 point mutation
    Authors: Marpaung, DSS;Jiang, SS;Fang, WT;Liao, YC;Chuang, MC
    Contributors: National Institute of Cancer Research;Institute of Population Health Sciences
    Abstract: The CRISPR-Cas12a-based diagnostic platform is renowned for its high sensitivity and, when coupled with the toehold-mediated strand displacement (TMSD) reaction, enables the detection of single nucleotide mismatch (SNM). However, conventional TMSD with the CRISPR-Cas12a system suffers from signal leakage due to overlapping activator sequences and low displacement efficiency. Here, we propose a toehold-embedded hairpinmediated strand displacement (THMSD) method and leverage the magnesium attenuation effect of CRISPRCas12a for selective single-point mutation detection in the TP53 gene. This approach effectively mitigates signal leakage as the hairpin DNA retains partial crRNA targeting sequences within its loop, preventing sequence overlap with the trigger and direct Cas12a activation. Additionally, the magnesium attenuation effect reduces background signal and enhances selectivity. We investigated toehold length and mismatch position to detect SNM with a high discrimination factor (DF). This high DF allows the THMSD/CRISPR-Cas12a system to differentiate between TP53 R273H mutant and TP53 wildtype genes for cancer screening at low abundances, down to 0.1 % with a concentration of 20 pM. When used for screening between H1975 and WI38 cells, the statistically significant difference was attainable with polymerase chain reaction (PCR) template loading as low as 4 ng. These results pave the way for developing CRISPR-Cas12a-based methods to effectively detect TP53 mutations as lung cancer biomarkers.
    Date: 2025-01-15
    Relation: Sensors and Actuators B-Chemical. 2025 Jan 15;423:Article number 136751.
    Link to: http://dx.doi.org/10.1016/j.snb.2024.136751
    Cited Times(WOS): https://www.webofscience.com/wos/woscc/full-record/WOS:001342664900001
    Cited Times(Scopus): https://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=85206243616
    Appears in Collections:[Shih-Sheng Jiang] Periodical Articles
    [Yu-Chieh Liao] Periodical Articles

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