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Detection of Ultralow-Frequency ctDNA Mutations Using a Dual Hairpin-Competition CRISPR/Cas14a System.
Zhaocheng Liu1, Rui Zhang1, Chunyan Chang1
1Department of Clinical Laboratory Medicine, The Affiliated Wuxi People's Hospital of Nanjing Medical University, Wuxi People's Hospital, Wuxi Medical Center, Nanjing Medical University, Wuxi 214023, China.
A new dual hairpin-competition CRISPR/Cas14a (DHCC) system significantly improves circulating tumor DNA (ctDNA) mutation detection sensitivity. This breakthrough enables earlier cancer detection and monitoring through highly accurate and affordable liquid biopsies.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Circulating tumor DNA (ctDNA) mutation profiling is crucial for cancer management.
- Current methods face limitations in sensitivity due to wild-type DNA interference, hindering clinical adoption.
Purpose of the Study:
- To develop a highly sensitive and specific platform for ctDNA mutation detection.
- To overcome the limitations of existing technologies for clinical liquid biopsy applications.
Main Methods:
- Introduction of a dual hairpin-competition CRISPR/Cas14a (DHCC) system.
- Integration of asymmetric PCR for mutant DNA enrichment and Cas14a detection with suppressed nonspecific binding.
- Utilized standard qPCR instruments for detection.
Main Results:
- Achieved a 58-fold improvement in mutant-wild-type discrimination (from 2.48 to 145).
- DHCC demonstrated a 250-fold sensitivity gain, detecting mutations at 0.002% variant allele frequency (VAF).
- Clinical validation showed 100% concordance with droplet digital PCR for EGFR L858R detection.
Conclusions:
- The DHCC system offers ultrahigh sensitivity and specificity for ctDNA analysis.
- DHCC provides a cost-effective and time-efficient alternative to current methods like ddPCR and NGS.
- This platform facilitates accessible ctDNA-based liquid biopsy for clinical settings.
