Li Xingrong, Wang Cuixiang, Chai Jiatong, Liu Hongmao, Jiang Xinli, Li Yumei, Li Yirong
Department of Laboratory Medicine, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, People's Republic of China.
Department of Laboratory Medicine, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, People's Republic of China; Wuhan Research Center for Infectious Diseases and Cancer, Chinese Academy of Medical Sciences, Wuhan, People's Republic of China; Hubei Engineering Center for Infectious Disease Prevention, Control and Treatment, Wuhan, People's Republic of China.
Anal Chim Acta. 2025 Jan 22;1336:343515. doi: 10.1016/j.aca.2024.343515. Epub 2024 Dec 3.
In current years, the CRISPR (clustered regularly interspaced short palindromic repeats) based strategies have emerged as the most promising molecular tool in the field of gene editing, intracellular imaging, transcriptional regulation and biosensing. However, the recent CRISPR-based diagnostic technologies still require the incorporation of other amplification strategies (such as polymerase chain reaction) to improve the cis/trans cleavage activity of Cas12a, which complicates the detection workflow and lack of a uniform compatible system to respond to the target in one pot.
To better fully-functioning CRISPR/Cas12a, we reported a novel technique for straightforward nucleic acid detection by incorporating enzyme-responsive steric hindrance-based branched inhibitors with CRISPR/AsCas12a methodology. The construction-transferable branched inhibitors coupled with a specific overhang flap induce spatial steric effects and result in the loss of the binding ability of Cas12a, which inhibits the activity of Cas12a. Target as the input signal would trigger the site-directed APE1 enzyme incision of the inhibitors, thus transforming the conformation of the inhibitors into split activators to illumine the CRISPR/AsCas12a catalyst system. At the same time, we found that APE1 could drive the enzymatic positive feedback circuit and exhibited considerably high amplification efficiency to enhance the detection ability of nucleic acids. Besides, our method provides universal platforms and can be realized in real-time and one-pot detection of HIV-1 DNA by replacing the inhibitors and crRNA with different target recognition sequences.
Overall, due to the high programmability of the nucleic acid network, this work proposed a feasible way to use the steric hindrance-based inhibitors as a switchable element, decorating the CRISPR/Cas12a-based strategy equipment for molecular diagnostics. Besides, this strategy could offer a simple tool for detecting trace nucleic acid, which opens avenues for future clinical application.
近年来，基于CRISPR（成簇规律间隔短回文重复序列）的策略已成为基因编辑、细胞内成像、转录调控和生物传感领域最有前景的分子工具。然而，最近基于CRISPR的诊断技术仍需要结合其他扩增策略（如聚合酶链反应）来提高Cas12a的顺式/反式切割活性，这使检测流程变得复杂，并且缺乏一个统一的兼容系统来在一锅反应中对靶标做出响应。
为了使CRISPR/Cas12a更好地发挥功能，我们报道了一种新技术，通过将基于酶响应空间位阻的分支抑制剂与CRISPR/AsCas12a方法相结合，用于直接核酸检测。可构建转移的分支抑制剂与特定的突出端侧翼相结合会诱导空间位阻效应，导致Cas12a结合能力丧失，从而抑制Cas12a的活性。作为输入信号的靶标会触发抑制剂的定点APE1酶切，从而将抑制剂的构象转变为裂解激活剂，以激活CRISPR/AsCas12a催化系统。同时，我们发现APE1可以驱动酶促正反馈回路，并表现出相当高的扩增效率，以增强核酸的检测能力。此外，我们的方法提供了通用平台，通过用不同的靶标识别序列替换抑制剂和crRNA，可以实现对HIV-1 DNA的实时一锅法检测。
总体而言，由于核酸网络的高度可编程性，这项工作提出了一种可行的方法，即使用基于空间位阻的抑制剂作为可切换元件，为基于CRISPR/Cas12a的分子诊断策略设备进行修饰。此外，该策略可以提供一种检测痕量核酸的简单工具，为未来的临床应用开辟了道路。
