Abstract：
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 Mg2+ 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.
摘要：
CRISPR/Cas13核酸酶已被广泛记录用于核酸检测。了解CRISPR/Cas13反应成分的复杂性对于充分发挥其在生物传感应用中的潜力至关重要。在这里，我们报告了CRISPR/Cas13a反应成分对其反式切割活性的影响，以及芯片上全内反射荧光显微镜（TIRFM）驱动的RNA传感系统的开发。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Mg2+浓度，记者长度,和crRNA组合显著提高了检测灵敏度。在优化条件下，我们使用微量滴定板读数器检测了100fM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传感工具箱，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100aM的RNA。此外，本研究中开发的基于TIRM的检测系统的灵敏度比下盖玻片测定法高1000倍.通过检测SARS-CoV-2假病毒RNA证明了该系统可能的临床适用性。我们提出的传感系统有可能检测任何靶RNA，对现有设置进行轻微修改，提供通用的RNA检测平台。
