Abstract 3672: High-sensitivity CRISPR-Cas12a detection of prostate-specific antigen based on computationally optimized aptamers

Abstract Background: Prostate cancer (PCa) is one of the most common malignant tumors worldwide. Early detection of PCa via Prostate-Specific Antigen (PSA) blood testing is essential for efficient and accurate screening. However, current diagnostic approaches are costly, labor-intensive, require large sample volumes and have relatively low interference capability. Additionally, the limitations of Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology often result in PSA aptamers with insufficient affinity, compromising the sensitivity of some aptamer-based detection methods. Methods: In this study, we developed a PSA detection strategy based on the trans-cleavage mechanism of the CRISPR-Cas12a-aptamer system, integrating computationally optimized PSA aptamers with CRISPR-Cas12a-based technology. A series of experiments were conducted, including the optimization of PSA aptamers, the design of crRNA, the optimization of aptamer concentration, the establishment of the CRISPR-Cas12a-aptamer detection system, and the validation of clinical samples. Results: Through sequence alignment and structural analysis of existing PSA aptamers, we identified conserved and potential binding sequences, enabling the construction of optimized aptamers. Molecular docking revealed aptamers with lower ΔG values and higher ZDOCK scores, which were selected as candidates. Optimal aptamer concentrations were identified experimentally, and the system's sensitivity and specificity were assessed using PSA standards and non-specific controls. Comparative studies demonstrated enhanced detection sensitivity with optimized aptamers, exhibiting a linear PSA detection range of 1.44-23 ng/ml with steeper slopes and higher R2 values compared to original aptamers. In the validation of clinical samples, the detection values of this method showed good consistency with the data provided by hospitals. Conclusions: This study demonstrates the ultrasensitivity and rapidity of the CRISPR-Cas12a-aptamer detection system and presents a robust strategy for improving aptamer affinity through sequence analysis and molecular docking, offering significant potential for diagnostic applications. Citation Format: Peizhi Ma, Jing Wu, Yichen Zhang, Fangyue Guo, Mengying Liu, Shuying Sun, Shize Xiao, Shanfeng Zhang, Pei Li. High-sensitivity CRISPR-Cas12a detection of prostate-specific antigen based on computationally optimized aptamers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3672.