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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.
Abstract:
Circulating tumor DNA (ctDNA) mutation profiling is essential for guiding targeted therapy and monitoring cancer recurrence, yet its clinical adoption is constrained by overwhelming wild-type DNA background and the limited sensitivity of existing platforms. Here, we introduce a dual hairpin-competition CRISPR/Cas14a (DHCC) system that integrates two sequential layers of hairpin competition: selective enrichment of mutant DNA during asymmetric PCR, followed by suppression of nonspecific sgRNA binding during Cas14a detection. This design dramatically enhances mutant-wild-type discrimination, elevating the discrimination factor from 2.48 to 145─a 58-fold improvement. While previous Cas14a methods achieve detection limits of 0.5-0.1% variant allele frequency (VAF), DHCC delivers a 250-fold sensitivity gain, routinely detecting four clinically relevant mutations (EGFR T790M, L858R, G719A, and NRAS Q61K) at VAFs as low as 0.002%. In multiplexed format, sensitivities of 0.005-0.01% VAF are maintained. Clinical validation using 22 plasma ctDNA samples demonstrated 100% concordance with droplet digital PCR for EGFR L858R detection. Compared to ddPCR and next-generation sequencing, DHCC substantially reduces turnaround time and cost while operating on standard qPCR instruments, eliminating the need for specialized infrastructure. By combining ultrahigh sensitivity, PAM independence, multiplexing preamplification capability, and practical affordability, DHCC provides an accessible platform for ctDNA-based liquid biopsy in clinical settings.
Insights
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.
