A mismatch-engineered crRNA PCR-CRISPR/Cas12a assay may improve detection of EGFR point mutations in single tumor cells.
Evidence
Platform experiment tested the assay with conical-pore membrane circulating tumor cell enrichment, reporting 0.1% mutation-frequency detection, 1.02 ng DNA sensitivity, and Kappa values above 0.9 versus NGS.
Caveat
The abstract presents analytical and tumor-cell detection performance, not clinical outcome evidence that the platform improves treatment decisions.
Simplified
Dynamic monitoring of epidermal growth factor receptor (EGFR) mutations is essential for the early identification of resistance and treatment adaptation. Single-cell heterogeneity analysis is crucial for precision cancer medicine, yet sensitive and specific detection methods for individual tumor cells remain challenging. Here, we develop a enhanced by the incorporation of mismatched base in crRNA at specific site for single-cell point mutation detection. This platform demonstrated high specificity and sensitivity, detecting point mutation at a frequency of 0.1% and in as low as 1.02 ng of genomic DNA, which represents an improvement over the amplification-refractory mutation system PCR (ARMS-PCR). Notably, the accuracy of the platform is highly consistent with next-generation sequencing (NGS), as evidenced by Kappa test values surpassing 0.9. By utilizing a conical-pore membrane with optimized porosity for single circulating tumor cell (CTC) enrichment, our platform enables point mutations detection in individual tumor cells, offering potential enhancements in precision and reliability for EGFR mutation analysis. This novel methodology holds potential for more accurate and personalized cancer treatment strategies.
Key numbers
0.1%
Detection Sensitivity
Sensitivity threshold for detecting .
3.76×
Increase
Increase in with -introduced .
0.9050
Kappa Test Value
Kappa test value for L858R mutation detection.
Full Text
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Declarations. Ethics approval: 43 tissue DNA samples of NSCLC were obtained from Sun Yat-sen University Cancer Center (SYSUCC). And the next-generation sequencing (NGS) was detected by the Department of Molecular Diagnostics of SYSUCC. The study protocol was approved by the Ethics Committee of SYSUCC (D2021-005). Competing interests: The authors declare no competing interests.
