SPEAR: CRISPR-mediated ultrasensitive, specific and rapid one-pot detection strategy for cancer-related SNPs.

Rationale: The ultrasensitive and accurate detection of driver mutations is critical for early cancer screening and precision medicine. Current methods face challenges in balancing sensitivity, specificity, and speed, which limits their clinical utility. Therefore, a rapid, sensitive, and specific method is essential for detecting cancer-related SNPs. Methods: This study introduces SPEAR (Specific Point mutation Evaluation via CRISPR-Cas Assisted Recognition), a novel methodology combining NEAR (Nicking Enzyme Amplification Reaction) isothermal amplification with SNP-specific recognition by Cas12b RNP in a one-pot configuration to detect cancer-related single nucleotide polymorphisms (SNPs). SPEAR leverages the power of NEAR isothermal amplification to efficiently amplify target DNA, followed by Cas12b RNP for SNP-specific recognition. This integrated approach ensures a rapid and precise mutation detection system in a single reaction. Results: The method was applied to blood samples for the detection of cancer-related mutations, with results obtained in approximately 30 min. The SPEAR enables detection of gene mutations at the single-molecule level and it can detect targets at a 0.1% ratio despite strong background interference. The method exhibits single-base resolution specificity, allowing for the detection of multiple SNPs in a single reaction. It outperforms first-generation sequencing (FGS) in both convenience and sensitivity, while remaining compatible with next-generation sequencing (NGS). Conclusion: SPEAR offers a rapid, sensitive, and convenient approach to detect cancer-related SNPs, with significant potential for clinical applications, including real-time detection and molecular diagnostics in precision medicine.