Yao Xia, Hu Xiaoyu, Wang Xiaoqi, Ge Jingyan
College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, Zhejiang, China.
Sheng Wu Gong Cheng Xue Bao. 2023 Jan 25;39(1):86-102. doi: 10.13345/j.cjb.220347.
Cell-free transcription and translation (TXTL) system is a cell extract-based system for rapid protein expression. The system bypasses routine laboratory processes such as bacterial transformation, clonal screening and cell lysis, which allows more precise and convenient control of reaction substrates, reduces the impact of bacteria on protein production, and provides a high degree of versatility and flexibility. In recent years, TXTL has been widely used as an emerging platform in clusterd regularly interspaced short palindromic repeat (CRISPR) technologies, enabling more rapid and convenient characterization of CRISPR/Cas systems, including screening highly specific gRNAs as well as anti-CRISPR proteins. Furthermore, TXTL-based CRISPR biosensors combined with biological materials and gene circuits are able to detect pathogens through validation of related antibiotics and nucleic acid-based markers, respectively. The reagents can be freeze-dried to improve portability and achieve point-of-care testing with high sensitivity. In addition, combinations of the sensor with programmable circuit elements and other technologies provide a non-biological alternative to whole-cell biosensors, which can improve biosafety and accelerate its application for approval. Here, this review discusses the TXTL-based characterization of CRISPR and their applications in biosensors, to facilitate the development of TXTL-based CRISPR/Cas systems in biosensors.
无细胞转录和翻译（TXTL）系统是一种基于细胞提取物的快速蛋白质表达系统。该系统绕过了诸如细菌转化、克隆筛选和细胞裂解等常规实验室流程，从而能够更精确、方便地控制反应底物，减少细菌对蛋白质生产的影响，并提供高度的通用性和灵活性。近年来，TXTL作为一种新兴平台已广泛应用于成簇规律间隔短回文重复序列（CRISPR）技术中，能够更快速、方便地表征CRISPR/Cas系统，包括筛选高特异性的引导RNA（gRNA）以及抗CRISPR蛋白。此外，基于TXTL的CRISPR生物传感器与生物材料和基因回路相结合，能够分别通过验证相关抗生素和基于核酸的标志物来检测病原体。这些试剂可以冻干以提高便携性，并实现高灵敏度的即时检测。此外，该传感器与可编程电路元件及其他技术的结合为全细胞生物传感器提供了一种非生物替代方案，可提高生物安全性并加速其获批应用。在此，本综述讨论了基于TXTL的CRISPR表征及其在生物传感器中的应用，以促进基于TXTL的CRISPR/Cas系统在生物传感器中的发展。
