School of Communication Engineering, Hangzhou Dianzi University, Hangzhou, 310018, Zhejiang, China.
Institute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen, 518055, Guangdong Province, China.
Mikrochim Acta. 2024 Jul 17;191(8):466. doi: 10.1007/s00604-024-06545-4.
The CRISPR/Cas13 nucleases have been widely documented for nucleic acid detection. Understanding the intricacies of CRISPR/Cas13's reaction components is pivotal for harnessing its full potential for biosensing applications. Herein, we report on the influence of CRISPR/Cas13a reaction components on its trans-cleavage activity and the development of an on-chip total internal reflection fluorescence microscopy (TIRFM)-powered RNA sensing system. We used SARS-CoV-2 synthetic RNA and pseudovirus as a model system. Our results show that optimizing Mg concentration, reporter length, and crRNA combination significantly improves the detection sensitivity. Under optimized conditions, we detected 100 fM unamplified SARS-CoV-2 synthetic RNA using a microtiter plate reader. To further improve sensitivity and provide a new amplification-free RNA sensing toolbox, we developed a TIRFM-based amplification-free RNA sensing system. We were able to detect RNA down to 100 aM. Furthermore, the TIRM-based detection system developed in this study is 1000-fold more sensitive than the off-coverslip assay. The possible clinical applicability of the system was demonstrated by detecting SARS-CoV-2 pseudovirus RNA. Our proposed sensing system has the potential to detect any target RNA with slight modifications to the existing setup, providing a universal RNA detection platform.
CRISPR/Cas13 核酸酶已被广泛用于核酸检测。了解 CRISPR/Cas13 的反应成分的复杂性对于充分利用其在生物传感应用中的潜力至关重要。在此，我们报告了 CRISPR/Cas13a 反应成分对其转切割活性的影响，并开发了一种基于片上全内反射荧光显微镜（TIRFM）的 RNA 传感系统。我们使用 SARS-CoV-2 合成 RNA 和假病毒作为模型系统。我们的结果表明，优化 Mg 浓度、报告分子长度和 crRNA 组合可以显著提高检测灵敏度。在优化条件下，我们使用微孔板读数仪检测到 100 fM 未扩增的 SARS-CoV-2 合成 RNA。为了进一步提高灵敏度并提供新的无扩增 RNA 传感工具包，我们开发了一种基于 TIRFM 的无扩增 RNA 传感系统。我们能够检测到低至 100 aM 的 RNA。此外，与盖玻片外检测相比，本研究中开发的基于 TIRM 的检测系统的灵敏度提高了 1000 倍。通过检测 SARS-CoV-2 假病毒 RNA，证明了该系统的潜在临床适用性。我们提出的传感系统通过对现有设置进行微小修改，有可能检测到任何目标 RNA，为通用的 RNA 检测平台提供了可能。
