Li Yueyuan, Hu Qianfang, Bai Meiqi, Qing Min, Bai Lijuan
Chongqing Research Center for Pharmaceutical Engineering, College of Pharmacy, Chongqing Medical University, Chongqing 400016, P. R. China.
Department of Respiratory and Critical Care Medicine, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, P. R. China.
Anal Chem. 2025 Feb 18;97(6):3617-3624. doi: 10.1021/acs.analchem.4c06107. Epub 2025 Feb 6.
Despite the widespread application of the CRISPR-Cas12a system in vitro diagnostics due to its high programmability and distinctive -cleavage activity, the susceptibility of its crRNA component to degradation and sensitivity to storage and working conditions poses a significant challenge to improving the practical efficacy of these diagnostic systems. Here, we show that engineered crRNA with a covalently closed circular structure (C-crRNA) can replace traditional linear crRNA to form functional complexes with Cas12a protein, significantly enhancing the anti-interference ability of the CRISPR-Cas12a system while maintaining its sensitivity and specificity. Based on this finding, a circular crRNA-mediated CRISPR molecular diagnostic (CRCD) toolkit is developed and successfully integrated with a standard nucleic acid amplification technique to detect synthesized Human Papillomavirus type 16 (HPV-16) plasmids down to 10 aM sensitivity levels. Furthermore, the CRCD system is applied for ultrasensitive detection of 40 HPV-16 and 40 influenza A viruses in clinical samples, with results consistent with those from PANTHER detection and quantitative real-time polymerase chain reaction (qRT-PCR). In conclusion, this strategy introduces a novel paradigm for engineering crRNA to program Cas12a, which has the potential to revolutionize the use of crRNA in CRISPR-based molecular diagnostics.
尽管CRISPR-Cas12a系统因其高度可编程性和独特的切割活性在体外诊断中得到广泛应用，但其crRNA组分易降解以及对储存和工作条件敏感，这对提高这些诊断系统的实际效能构成了重大挑战。在此，我们表明具有共价闭合环状结构的工程化crRNA（C-crRNA）可以取代传统的线性crRNA与Cas12a蛋白形成功能复合物，在保持其敏感性和特异性的同时显著增强CRISPR-Cas12a系统的抗干扰能力。基于这一发现，开发了一种环状crRNA介导的CRISPR分子诊断（CRCD）工具包，并成功与标准核酸扩增技术整合，以检测合成的人乳头瘤病毒16型（HPV-16）质粒，检测灵敏度低至10 aM。此外，CRCD系统用于临床样本中40种HPV-16和40种甲型流感病毒的超灵敏检测，结果与PANTHER检测和定量实时聚合酶链反应（qRT-PCR）的结果一致。总之，该策略引入了一种工程化crRNA以编程Cas12a的新范式，这有可能彻底改变crRNA在基于CRISPR的分子诊断中的应用。
