In addition to their roles as revolutionary genome engineering tools, CRISPR-Cas systems are also highly promising candidates in the construction of biosensing systems and diagnostic devices, which have attracted significant attention recently. However, the CRISPR-Cas system cannot be directly applied in the sensing of non-nucleic acid targets, and the needs of synthesizing and storing different vulnerable guide RNA for different targets also increase the application and storage costs of relevant biosensing systems, and therefore restrict their widespread applications. To tackle these barriers, in this work, a versatile CRISPR-Cas12a-based biosensing platform was developed through the introduction of an enzyme-free and robust DNA reaction network, the entropy-driven dynamic DNA network. By programming the sequences of the system, the entropy-driven catalysis-based dynamic DNA network can respond to different types of targets, such as nucleic acids or proteins, and then activate the CRISPR-Cas12a to generate amplified signals. As a proof of concept, both nucleic acid targets (a DNA target with random sequence, T, and an RNA target, microRNA-21 (miR-21)) and a non-nucleic acid target (a protein target, thrombin) were chosen as model analytes to address the feasibility of the designed sensing platform, with detection limits at the pM level for the nucleic acid analytes (7.4 pM for the DNA target T and 25.5 pM for miR-21) and 0.4 nM for thrombin. In addition, the detection of miR-21 or thrombin in human serum samples further demonstrated the applicability of the proposed biosensing platform in real sample analysis.

中文翻译：

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多功能基于 CRISPR-Cas12a 的生物传感平台，采用可编程熵驱动动态 DNA 网络进行调制

除了作为革命性基因组工程工具的作用外，CRISPR-Cas 系统在生物传感系统和诊断设备的构建中也是非常有前途的候选者，最近引起了广泛关注。然而，CRISPR-Cas系统不能直接应用于非核​​酸靶点的传感，针对不同靶点需要合成和储存不同脆弱的向导RNA，也增加了相关生物传感系统的应用和储存成本，因此限制它们的广泛应用。为了解决这些障碍，在这项工作中，通过引入无酶且稳健的 DNA 反应网络，即熵驱动的动态 DNA 网络，开发了一种基于 CRISPR-Cas12a 的多功能生物传感平台。通过对系统的序列进行编程，熵驱动的基于催化的动态 DNA 网络可以响应不同类型的目标，例如核酸或蛋白质，然后激活 CRISPR-Cas12a 以产生放大信号。作为概念证明，两种核酸靶标（具有随机序列的 DNA 靶标，T和 RNA 靶标 microRNA-21 (miR-21)) 和非核酸靶标（蛋白质靶标，凝血酶）被选为模型分析物，以解决设计传感平台的可行性，检测限在核酸分析物的 pM 水平（DNA 靶标T为 7.4 pM，miR-21 为 25.5 pM），凝血酶为 0.4 nM。此外，人血清样品中 miR-21 或凝血酶的检测进一步证明了所提出的生物传感平台在实际样品分析中的适用性。