Zhang Yuan, Yu Wanpeng, Wang Man, Zhang Lei, Li Peifeng
Institute for Translational Medicine, The Affiliated Hospital of Qingdao University, Qingdao University, Qingdao, China.
Medical Collage, Qingdao University, Qingdao, China.
Front Bioeng Biotechnol. 2024 Jan 5;11:1327498. doi: 10.3389/fbioe.2023.1327498. eCollection 2023.
The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR associated) system has proven to be a powerful tool for nucleic acid detection due to its inherent advantages of effective nucleic acid identification and editing capabilities, and is therefore known as the next-generation of molecular diagnostic technology. However, the detection technologies based on CRISPR/Cas systems require preamplification of target analytes; that is, target gene amplification steps through isothermal amplification or PCR before detection to increase target analyte concentrations. This creates a number of testing limitations, such as extended testing time and the need for more sophisticated testing instruments. To overcome the above limitations, various amplification-free assay strategies based on CRISPR/Cas systems have been explored as alternatives, which omit the preamplification step to increase the concentrations of the target analytes. Nanozymes play a pivotal role in enhancing the sensitivity of CRISPR-based detection, enabling visual and rapid CRISPR assays. The utilization of nanozyme exceptional enzyme-like catalytic activity holds great promise for signal amplification in both electrochemical and optical domains, encompassing strategies for electrochemical signal sensors and colorimetric signal sensors. Rather than relying on converting a single detection target analyte into multiple analytes, these methods focus on signal amplification, the main mechanism of which involves the ability to form a large number of reporter molecules or to improve the performance of the sensor. This exploitation of nanozymes for signal amplification results in the heightened sensitivity and accuracy of detection outcomes. In addition to the strategies that improve sensor performance through the application of nanozymes, additional methods are needed to achieve visual signal amplification strategies without preamplification processes. Herein, we review the strategies for improving CRISPR/Cas systems that do not require preamplification, providing a simple, intuitive and preamplification-free CRISPR/Cas system detection platform by improving in-system one-step amplification programs, or enhancing nanozyme-mediated signal amplification strategies.
CRISPR（成簇规律间隔短回文重复序列）/Cas（CRISPR相关）系统因其在核酸识别和编辑能力方面的固有优势，已被证明是一种强大的核酸检测工具，因此被誉为下一代分子诊断技术。然而，基于CRISPR/Cas系统的检测技术需要对目标分析物进行预扩增；也就是说，在检测前通过等温扩增或聚合酶链反应（PCR）进行目标基因扩增步骤，以提高目标分析物的浓度。这带来了许多检测限制，如检测时间延长以及需要更复杂的检测仪器。为克服上述限制，人们探索了各种基于CRISPR/Cas系统的无扩增检测策略作为替代方案，这些策略省略了预扩增步骤以增加目标分析物的浓度。纳米酶在提高基于CRISPR的检测灵敏度方面发挥着关键作用，可实现可视化和快速的CRISPR检测。利用纳米酶独特的类酶催化活性在电化学和光学领域的信号放大方面具有巨大潜力，包括电化学信号传感器和比色信号传感器策略。这些方法并非依赖于将单个检测目标分析物转化为多个分析物，而是专注于信号放大，其主要机制涉及形成大量报告分子的能力或改善传感器的性能。这种利用纳米酶进行信号放大可提高检测结果的灵敏度和准确性。除了通过应用纳米酶改善传感器性能的策略外，还需要其他方法来实现无需预扩增过程的可视化信号放大策略。在此，我们综述了改进CRISPR/Cas系统的无需预扩增的策略，通过改进系统内一步扩增程序或增强纳米酶介导的信号放大策略，提供一个简单、直观且无需预扩增的CRISPR/Cas系统检测平台。
