Work

Detection of Ultralow-Frequency ctDNA Mutations Using a Dual Hairpin-Competition CRISPR/Cas14a System

Year: 2026

Type: article

Abstract: High Resolution Image Download MS PowerPoint Slide 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.

Source: Analytical Chemistry

DOI: 10.1021/acs.analchem.6c02495