Enhanced SNV Detection of HLA-B*15:02TA by Integrating Blocking RPA and Inhibiting CRISPR-Cas12a.

With the introduction of the RPA-based CRISPR-Cas12a method, crRNA allostery and Cas12a protein engineering have been applied to the identification of single-nucleotide variants (SNVs). However, the complicated testing procedure often falls short of the intended simple and rapid objectives of CRISPR-based diagnostics (CRISPR-Dx), and the discrimination factor (DF) for SNV distinction remains suboptimal (DF = 2-5). In this study, we proposed a one-tube "Blocking RPA-coupled Inhibiting CRISPR-Cas12a" (BRIC) cascade strategy that features a dual recognition of SNVs. In the BRIC cascade, a reduction of RPA amplification products of wild-type target (WT) and WT-activated Cas12a cleavage lowered the WT detection signal to near-background levels, without compromising the efficiency of RPA or CRISPR-Cas12a. With HLA-B*15:02TA (rs3909184, G > C), a gene associated with drug-induced cutaneous rash, as the SNV target, the proposed one-tube BRIC strategy achieved a DF of 32.51, with the WT signal approximating the background level, thereby greatly enhancing SNV detection specificity. On the other hand, MT can be detected in a large number of WT (with a resolution of 0.1%). Within the HLA-B*15:02TA concentration (CMT) range of 5.0 × 10-7 to 1.0 × 10-3 nM (500 aM to 1 pM), a linear relationship was evident between the fluorescence signal (FMT) and lgCMT (FMT = 4146.55 + 578.09lgCMT; R2 = 0.9904), with a detection limit of 67.6 aM (>3σ). Combined with a 3D-printed portable fluorimeter, the proposed method reported high consistency with next-generation sequencing in detecting HLA-B*15:02TA in 22 clinical plasma samples, validating the reliability of the one-tube BRIC cascade assay. These improvements in assay design, process, and DF highlight the promising potentials of the BRIC strategy in clinical settings.