Pan Yongchun, Luan Xiaowei, Zeng Fei, Xu Qin, Li Zekun, Gao Yanfeng, Liu Xinli, Li Xueqing, Han Xin, Shen Jianliang, Song Yujun
College of Engineering and Applied Sciences, State Key Laboratory of Analytical Chemistry for Life Science, Nanjing University, Nanjing, 210023, China; Engineering Research Center of Clinical Functional Materials and Diagnosis & Treatment Devices of Zhejiang Province, Wenzhou Institute, University of Chinese Academy of Sciences, Wenzhou, 325024, China.
College of Engineering and Applied Sciences, State Key Laboratory of Analytical Chemistry for Life Science, Nanjing University, Nanjing, 210023, China.
Biosens Bioelectron. 2022 Aug 1;209:114239. doi: 10.1016/j.bios.2022.114239. Epub 2022 Apr 3.
In addition to applications in genome editing, clustered regularly interspaced short palindromic repeats (CRISPR) have recently been engineered for medical diagnostics based on their trans-cleavage activity owing to their high base resolution and isothermal signal amplification. However, trans-cleavage activity is too fragile to be applied in vivo. Herein, we introduce a hollow covalent organic framework (COF)-sheltering CRISPR/aptamer-based sensor (h-CCS) for ATP imaging in living animals. The CRISPR/aptamer-based complex is comprised of the CRISPR-Cas12a system, fluorophore quencher-labeled single-stranded DNA substrate (ssDNA-FQ), and a DNA activator that pre-hybridizes with ATP aptamer to prevent the trans-cleavage activity of the Cas12a system in the absence of ATP. After being encapsulated in a hollow COF, the constructed nanoreactor is highly robust and can be lit up by ATP for in vivo imaging. Considering the unique properties of h-CCS, this strategy offers great potential to broaden applications of not only CRISPR-Cas systems but also other proteins in porous matrixes for clinical diagnostics, medical research, and biomimetic nanodevices.
除了在基因组编辑中的应用外，成簇规律间隔短回文重复序列（CRISPR）最近还因其高碱基分辨率和等温信号放大的反式切割活性而被设计用于医学诊断。然而，反式切割活性过于脆弱，无法在体内应用。在此，我们介绍一种用于活体动物ATP成像的中空共价有机框架（COF）保护的基于CRISPR/适体的传感器（h-CCS）。基于CRISPR/适体的复合物由CRISPR-Cas12a系统、荧光团淬灭剂标记的单链DNA底物（ssDNA-FQ）和一种与ATP适体预杂交以在没有ATP时防止Cas12a系统反式切割活性的DNA激活剂组成。封装在中空COF中后，构建的纳米反应器具有很高的稳定性，并且可以被ATP点亮用于体内成像。考虑到h-CCS的独特性质，这种策略不仅为拓宽CRISPR-Cas系统的应用，也为拓宽其他蛋白质在多孔基质中用于临床诊断、医学研究和仿生纳米器件的应用提供了巨大潜力。
