Research Center of Pharmacology and Toxicology, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China.
Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, College of Future Technology, and State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing, China.
Commun Biol. 2024 Oct 12;7(1):1312. doi: 10.1038/s42003-024-07000-z.
Cas12 and Cas13 are extensively utilized in molecular diagnostics for their trans-cleavage activities, yet their activation characteristics remain partially understood. Here, we conduct an in-depth investigation of Cas12a, Cas12f1, and Cas13a, uncovering the characteristics of their trans-DNase and trans-RNase activities with noncanonical activators. Our findings reveal that DNA can serve as a direct target for CRISPR-Cas13a, markedly increasing the detection sensitivity for single-base mismatches. Moreover, the trans-cleavage activities of Cas12a and Cas13a can be activated by diverse RNA:DNA and RNA:RNA duplexes, respectively, indicating that the presence of stem-loop structures in crRNAs is not essential for their activation. Notably, Cas12f1, unlike Cas12a, exhibits intrinsic RNase activity independently of activation. Leveraging these insights, we have improved the accuracy of a dual-gene target detection approach that employs the CRISPR-Cas12f1 and Cas13a systems. Our research advances the understanding of the noncanonical activation characteristics of Cas12 and Cas13a, contributing to the field of CRISPR-based diagnostics.
Cas12 和 Cas13 因其转切割活性而被广泛应用于分子诊断，但它们的激活特性仍部分未知。在这里，我们深入研究了 Cas12a、Cas12f1 和 Cas13a，揭示了它们在非经典激活剂作用下的转 DNA 酶和转 RNA 酶活性的特征。我们的研究结果表明，DNA 可以作为 Cas13a 的直接靶标，显著提高对单碱基错配的检测灵敏度。此外，Cas12a 和 Cas13a 的转切割活性可分别被不同的 RNA:DNA 和 RNA:RNA 双链体激活，这表明 crRNA 中茎环结构的存在对其激活并非必需。值得注意的是，Cas12f1 与 Cas12a 不同，其内在的 RNase 活性无需激活即可发挥作用。利用这些见解，我们提高了一种双基因靶标检测方法的准确性，该方法同时使用了 Cas12f1 和 Cas13a 系统。我们的研究推进了对 Cas12 和 Cas13a 的非经典激活特性的理解，为基于 CRISPR 的诊断领域做出了贡献。
