An Optimized CRISPR/Cas12a Assay to Facilitate the BRAF V600E Mutation Detection

Background: Accurate detection of the BRAF V600E (1799T > A) mutation status can significantly contribute to selecting an optimal therapeutic strategy for diverse cancer types. CRISPR-based diagnostic platforms exhibit simple programming, cost-effectiveness, high sensitivity, and high specificity in detecting target sequences. The goal of this study is to develop a simple BRAF V600E mutation detection method. Methods: We combined the CRISPR/Cas12a system with recombinase polymerase amplification (RPA). Subsequently, several parameters related to CRISPR/Cas12a reaction efficiency were evaluated. Then, we conducted a comparative analysis of three distinct approaches toward identifying BRAF V600E mutations in the clinical samples. Results: Our data suggest that CRISPR/Cas detection is considerably responsive to variations in buffer conditions. Magnesium acetate (MgOAc) demonstrated superior performance compared to all other examined additive salts. It was observed using 150 nM guide RNA (gRNA) in an optimized reaction buffer containing 14 mM MgOAc, coupled with a reduction in the volumes of PCR and RPA products to 1 mu L and 3 mu L, respectively, resulted in an enhanced sensitivity. Detection time was decreased to 75 min with a 2% limit of detection (LOD), as evidenced by the results obtained from the blue light illuminator. The CRISPR/Cas12a assay confirmed the real-time PCR results in 31 of 32 clinical samples to identify the BRAF V600E mutation status, while Sanger sequencing detected BRAF V600E mutations with lower sensitivity. Conclusion: We propose a potential diagnostic approach that is facile, fast, and affordable with high fidelity. This method can detect BRAF V600E mutation with a 2% LOD without the need for a thermocycler.