Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, PR China.
Chongqing Key Laboratory of Sichuan-Chongqing Co-construction for Diagnosis and Treatment of Infectious Diseases Integrated Traditional Chinese and Western Medicine, Chongqing Hospital of Traditional Chinese Medicine, Chongqing 400021, PR China.
J Biotechnol. 2024 Aug 10;391:99-105. doi: 10.1016/j.jbiotec.2024.06.011. Epub 2024 Jun 14.
The CRISPR/Cas12a system is emerging as a promising candidate for next-generation diagnostic biosensing platforms, with the discovery of new activation modes greatly expanding its applications. Here, we have identified two novel CRISPR/Cas12a system activation modes: PAM- and toehold-free DNA hairpins, and DNA-RNA hybrid strands. Utilizing a well-established real-time fluorescence method, we have demonstrated a strong correlation between DNA hairpin structures and Cas12a activation. Compared with previously reported activation modes involving single-stranded DNA and PAM-contained double-stranded DNA, the DNA hairpin activation way exhibits similar specificity and generality. Moreover, our findings indicate that increasing the number of RNA bases in DNA-RNA hybrid strands can decelerate the kinetics of Cas12a-triggered trans-cleavage of reporter probes. These newly discovered CRISPR/Cas12a activation ways hold significant potential for the development of high-performance biosensing strategies.
CRISPR/Cas12a 系统作为下一代诊断生物传感平台的候选者正在兴起，新的激活模式的发现极大地扩展了其应用。在这里，我们已经确定了两种新的 CRISPR/Cas12a 系统激活模式：无 PAM 和无结合臂 DNA 发夹，以及 DNA-RNA 杂交链。利用成熟的实时荧光法，我们已经证明了 DNA 发夹结构与 Cas12a 激活之间存在很强的相关性。与以前报道的涉及单链 DNA 和含有 PAM 的双链 DNA 的激活模式相比，DNA 发夹激活方式具有相似的特异性和通用性。此外，我们的研究结果表明，增加 DNA-RNA 杂交链中的 RNA 碱基数量可以减缓 Cas12a 触发报告探针的反式切割的动力学。这些新发现的 CRISPR/Cas12a 激活方式为开发高性能生物传感策略提供了巨大的潜力。
