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Single-nucleotide variant profiling in liquid biopsy with RECO-Cas.
Chong Guo1,2, Shuo Zhang1,2, Ramachandra Yerramsetti1
1Department of Biomedical Engineering, University of Connecticut Health Center, Farmington, CT 06030, USA.
A new RECO-Cas assay detects cell-free DNA (cfDNA) mutations with high accuracy. This rapid, cost-effective liquid biopsy method aids precision oncology and early cancer diagnosis.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Liquid biopsy via cell-free DNA (cfDNA) mutation detection is crucial for precision oncology.
- Current methods lack simplicity, speed, accuracy, and cost-effectiveness, limiting clinical use.
Purpose of the Study:
- To develop a simple, rapid, accurate, and cost-effective assay for cfDNA mutation profiling.
- To enable sensitive detection of low-frequency mutations for clinical applications.
Main Methods:
- Developed the recombined DNA construct-activated Cas12a (RECO-Cas) assay.
- Utilized Argonaute-nicked mutant DNA and an artificial DNA activator to trigger CRISPR-Cas12a.
- Detected point mutations (KRAS, EGFR, PIK3CA) in cfDNA from clinical plasma samples.
Main Results:
- Achieved 0.01% variant allele frequency sensitivity and single-nucleotide resolution.
- Demonstrated high sensitivity (90.48%) and 100% specificity in detecting cfDNA mutations.
- Successfully classified and identified KRAS missense variants.
Conclusions:
- RECO-Cas assay offers a simple, rapid, and affordable solution for cfDNA mutation detection.
- The assay is compatible with point-of-care diagnostics using smartphone-based fluorescence detection.
- Enables sensitive detection of low-frequency mutations, supporting early cancer diagnosis and personalized treatment.
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