一种基于CRISPR/Cas14a的平台，通过短单链DNA扩增和分割目标设计，实现对单核苷酸变异的高灵敏度和高特异性检测。

Abstract

SAST enhances specificity through split target design while achieving high sensitivity via isothermal amplification. • For SNV detection, the SAST assay achieves a Concentration LOD of 100 aM for target DNA and a MAF LOD of 0.00001% in simulated samples. • SAST assay was successfully validated for SNV detection in tumor samples and pharmacogenetic genotyping. Single-nucleotide variant (SNV) detection, including tumor-associated mutation detection and pharmacogenetic genotyping, holds significant clinical value. Among current methods, CRISPR/Cas14a-based approaches offer advantages in specificity, sensitivity, and efficiency. However, the inherent single-stranded DNA (ssDNA) targeting property of Cas14a limits its direct application to genomic DNA detection, and its specificity requires further optimization to meet clinical standards. Here, we developed a CRISPR/Cas14a system based on s hort ssDNA a mplification and s plit t arget design (SAST), which substantially enhances specificity through split target design while achieving high sensitivity via short ssDNA amplification. Experimental results demonstrate that SAST system achieves a Concentration LOD (limit of detection) of 100 aM for target DNA and a MAF (mutant allele frequency) LOD of 0.00001% in simulated samples. The platform was successfully validated for SNV detection in tumor samples (KRAS G12D, rs121913529, c.35G > A) and pharmacogenetic genotyping (CYP2C19*2, rs4244285, c.681G > A; CYP2C19*3, rs4986893, c.636G > A; and CYP2C19*17, rs12248560, c.806C > T). SAST system offers a novel method for clinical single-base mutation detection and is expected to find broad applications in early cancer diagnosis and precision medicine. Download: Download high-res image (269KB) Download: Download full-size image