Rationale As a recognized diagnostic marker for prostate cancer, prostate-specific antigen (PSA) in human serum is important for early detection of prostate cancer. Based on the trans-cleavage principle of the CRISPR-Cas12a system, we developed a quantitative and qualitative assay for PSA using PSA-aptamer (PSA-APT). The system consists of Cas12a, crRNA, ssDNA probe and PSA-APT. When PSA is present in the system, PSA-APT preferentially binds to PSA, and PSA-APT bound to crRNA decreases, resulting in a reduction of the Cas12a trans-cut F-Q probe and a decrease in fluorescence intensity. Design The aptamers were optimized and the corresponding crRNAs were designed, and the system components were optimized to establish a quantitative fluorescence detection system and a qualitative CRISPR-combined lateral flow chromatography (CRISPR-LFA) detection system, then sensitivity and specificity experiments were performed and clinical samples were tested. Experimental contents ① Optimization for better performing aptamers through multiple sequence alignment, molecular docking, and experimental validation. ② Designing crRNAs that bind to aptamers and experimentally screening for better performing crRNAs. ③ Optimization of aptamer concentration, aptamer-PSA binding environment, CRISPR-Cas12a system components, etc. ④ CRISPR-LFA qualitative detection of PSA was achieved by optimizing the concentrations of the FITC-biotin probe, aptamer, and colloidal gold labeled antibody. ⑤ Sensitivity and specificity assays were performed using gradient standards and interferents such as CEA and AFP. ⑥ Qualitative and quantitative tests were performed on 483 clinical samples to demonstrate the validity of the above two testing systems. Materials PSA-APT, crRNA, PSA standard, Cas12a enzyme, LFA-related materials, etc. Preliminary experimental results ① The molecular docking fraction of the optimized aptamer was greatly improved, as evidenced by its ability to activate Cas12a trans-cutting activity as well as system sensitivity. ② Successful optimizing to get a better crRNA. ③ Improved sensitivity through system optimization. ④ Preliminary establishment of fluorescence quantification and CRISPR-LFA qualitative detection system. ⑤ The detection system can specifically detect PSA. ⑥ The detection system initially realizes qualitative and quantitative detection of clinical samples. ⑦ The ROC curve verifies that this method is comparable to existing methods in both qualitative CRISPR-LFA detection and quantitative fluorescence detection. Feasibility The CRISPR-Cas12a-aptamer detection system is technically sound, and the system has been validated by pre-experimental results, and initially realizes fluorescence quantitative detection of PSA and CRISPR-LFA qualitative detection, of which the CRISPR-LFA qualitative detection has already formed a preliminary product. Innovation In this study, we have established a method for rapid ultrasensitive quantitative and qualitative detection of PSA by CRISPR-Cas12a-aptamer system, and successfully used multiple sequence alignment combined with molecular docking to optimize a PSA aptamer with better performance, which promotes cross-fertilization of medicine and various disciplines, and is expected to lead the product to engineering and mass production.

Keywords: Aptamer; CRISPR-Cas12a; CRISPR-LFA; PSA; Detection

Zhang Shanfeng, male, is a senior experimentalist and master's supervisor. He is a member of the Microbiology Society of Henan Province, an active member of the American Association for Cancer Research (AACR), and a lifelong member of the Chinese Society of Biochemistry and Molecular Biology. He serves as an editorial board member for the Journal of Surgery and Implants and the Online Journal of Cardiology Research & Reports (OJCRR), and as a reviewer for Frontiers, Anti-Cancer Agents in Medicinal Chemistry, Current Chemical Biology, and Life Science and Technology. He has published more than 36 SCI papers with a total citation of 821. He has undertaken 5 projects funded by the National Natural Science Foundation of China, and has edited 4 textbooks and monographs. He has obtained 4 utility model patents. He has received the Second Prize of Scientific and Technological Progress in Henan Province and the Special Prize of Higher Education Teaching Achievement Award in Henan Province.

Memberships:

Microbiology Society of Henan Province

American Association for Cancer Research (AACR)

Chinese Society of Biochemistry and Molecular Biology

Editorial Board Member:

Journal of Surgery and Implants

Online Journal of Cardiology Research & Reports (OJCRR)

Reviewer for Journals:

Frontiers

Anti-Cancer Agents in Medicinal Chemistry

Current Chemical Biology

Life Science and Technology

Publications:

More than 36 SCI papers with a total citation of 821

Research Projects:

Undertaken 5 projects funded by the National Natural Science Foundation of China

Books and Monographs:

Editor of 4 textbooks and monographs

Patents:

Obtained 4 utility model patents

Awards:

Second Prize of Scientific and Technological Progress in Henan Province

Special Prize of Higher Education Teaching Achievement Award in Henan Province