Double-end blocker and split input mediated CRISPR-Cas12a system for modular single-base mutation detection with low abundance.

Abstract

Traditional CRISPR-Cas12a mutation detection systems are limited by poor single-base specificity, target-specific crRNA redesign, and insufficient sensitivity for low-abundance mutations, restricting their clinical liquid biopsy applications. Herein, we developed a crRNA-universal, sensitive and specific CRISPR-Cas12a detection platform, termed DESIC (double-end blocker and split-input mediated CRISPR-Cas12a system), for single-base mutation detection. The DESIC system adopts two key structural designs: double-end blocker (DEB) and duplicated split-input (SIN). The DEB spatially isolates crRNA recognition and target-binding regions, enabling universal detection of various mutation sites without crRNA redesign. The SIN strategy amplifies thermodynamic differences from single-base mismatches, greatly improving single-nucleotide discrimination. We targeted four prevalent pancreatic cancer KRAS mutations (G12D, G12R, G12V, Q61H) and optimized the system to achieve optimal discrimination. The optimized DESIC system exhibited ultra-low limits of detection down to 0.01% mutant allele fraction with reliable linear quantitative performance. Clinical validation using 15 pairs of pancreatic cancer tissue and peripheral blood samples confirmed that DESIC results were highly consistent with gold-standard NGS data. With a flexible modular design, this low-cost, easy-operated platform can be readily extended to multiple tumor mutations, holding great potential for tumor liquid biopsy and early molecular diagnosis.