基于邻位PAM组装的CRISPR/Cas12a通用型检测系统的构建及其应用研究

核酸和蛋白质的高灵敏检测对于生命科学研究和疾病的早期诊断具有重要意义。传统检测技术因灵敏度低、操作程序复杂、成本昂贵，无法满足科研或临床实验室的需求。CRISPR/Cas12a检测系统具有简单、快速、灵敏、廉价等优势，但由于受到PAM识别位点的限制，难以在核酸和蛋白检测中广泛应用。本项目在前期工作基础上，拟通过探索PAM位点的识别机制，对CRISPR/Cas12a系统进行改造和优化，克服PAM位点的依赖；结合邻位诱导DNA组装技术，实现高效的靶标识别与信号转化，建立一种简单快速、性能稳定、成本低廉，可广泛用于复杂实际样本中低丰度核酸和蛋白质高灵敏、高特异性检测的通用型检测系统。并将该方法初步应用于前列腺癌患者血清PSA的检测，验证其临床诊断性能。本项目的实施将为生命科学研究和临床实验诊断提供有力的分析工具；同时为充分开发CRISPR/Cas系统的应用潜能提供新的思路和实验基础。

The sensitive detection of nucleic acids and proteins is of great significance for life science research and early diagnosis of diseases. Traditional detection techniques cannot meet the needs of scientific research or clinical laboratory because of their low sensitivity, complicated operation procedures and high cost. The CRISPR/Cas12a detection system has the advantages of simple, fast, sensitive and cheap, but it is difficult to be widely used in nucleic acid and protein detection due to the limitation of PAM recognition sites. On the basis of the previous work, this project intends to transform and optimize the CRISPR/Cas12a system by exploring the recognition mechanism of PAM locus to overcome the dependence of PAM sequence. With the combination of proximity-induced DNA assembly technology, efficient target recognition and signal transformation can be achieved, and a simple, fast, stable and low-cost universal detection system can be developed for the detection of low abundance nucleic acids and proteins in complex samples with high sensitivity and specificity. The method was applied to the detection of serum PSA in prostate cancer patients to verify its clinical diagnostic performance. The implementation of this project will provide a powerful analytical tool for life science research and clinical trial diagnosis. At the same time, it provides new ideas and experimental basis for fully developing the application potential of CRISPR/Cas system.