Abstract
Pathogenic germline variants in the BRCA1 and BRCA2 tumor suppressor genes markedly increase lifetime risks of developing hereditary breast and ovarian cancer. While high-throughput next-generation sequencing serves as the gold standard for clinical profiling, its reliance on centralized laboratory infrastructure, prolonged turnaround times, and high cost restrict its utility in decentralized and resource-constrained settings. In this study, we developed an integrated bioinformatics and molecular diagnostic pipeline utilizing CRISPR-Cas12a coupled with recombinase polymerase amplification (RPA) for the rapid, point-of-care detection of founder pathogenic variants in BRCA1 (c.68_69delAG and c.5266dupC) and BRCA2 (c.5946delT). Using an in silico design framework that optimizes CRISPR RNAs (crRNAs) based on secondary structure stability, protospacer-adjacent motif positioning, and predicted off-target free energy minimization, we engineered specific discrimination assays capable of distinguishing single-nucleotide insertions and deletions. The assay couples isothermal pre-amplification with Cas12a trans-cleavage activation of quenched fluorescent and lateral-flow reporters, achieving analytical sensitivity down to 10 copies per microliter within 45 minutes at 37 °C. Benchmarking against validated clinical genomic DNA samples demonstrated 100% concordance with Sanger sequencing without requiring thermal cycling. This translational platform establishes an accessible, highly accurate modality for on-site cancer risk stratification, demonstrating how computational design and CRISPR collateral cleavage synergize to democratize clinical genomics.
