CRISPR/Cas12a-calibrated biosensor for accurate detection of low-frequency single nucleotide variants via loop-assisted adjustable entropy-driven catalytic reaction

Abstract

Accurate determination of single nucleotide variants (SNVs) with low variant-allele frequencies (VAFs) in clinical samples is urgently needed but challenging, since subtle Gibbs free energy differences of mutation-type (MT) sequences cannot overwhelm the interferences caused by high concentrations of wild-type (WT) sequences. Herein, a novel biosensor was constructed for accurately identifying SNVs with low VAFs (< 0.1 %) by a loop-assisted adjustable entropy-driven catalytic reaction (EDR), benefiting from the adjusted Gibbs free energy for these interferences elimination. At the detection system, the binding energy between WT sequences and EDR induced is restricted through the insertion of a loop structure in the toehold region, reducing the interferences generated by high concentrations of WT sequences. Furthermore, the CRISPR/Cas12a system functions as a synergistic signal calibrator, thereby markedly enhancing the sensitivity and specificity of the EDR through its precise RNA-guided nucleic acid recognition and signal amplification capability. Moreover, our proposed method was applied for drug-resistant mutations of Mycobacterium tuberculosis analysis from synthetic DNA and clinical samples, and excellent accuracy indicated its great potential as next-generation sequencing (NGS). This work provides some new insights about highly specific bioassays construction in disease diagnosis.