无细胞系统在CRISPR技术和生物传感器中的应用研究进展

Application of cell-free transcription and translation system in CRISPR technologies and the associated biosensors

摘要:

无细胞转录翻译系统(cell-free transcription and translation, TXTL)，简称无细胞系统，即基于细胞提取物在体外快速表达蛋白质的系统。该系统绕过了细菌转化、克隆筛选和细胞裂解等过程，更精准便捷地控制反应底物，减少细菌对蛋白质产生的影响，具有多功能性和灵活性等优点。近年来，TXTL作为一个新兴平台在clusterd regularly interspaced short palindromic repeat (CRISPR)技术研究中应用广泛，实现了对CRISPR/Cas系统快速、便捷地表征，比如高特异性的向导RNA (guide RNA, gRNA)以及抗CRISPR蛋白的筛选。同时，基于TXTL的CRISPR生物传感器与生物材料、基因回路等结合，开发并用于病原体中核酸标志物等的检测，相关试剂冻干后也提高了便携性，实现高灵敏度的即时检测(point-of-care testing, POCT)。传感器与可编程的电路元件等技术的结合为全细胞生物传感器(whole cell biosensor, WCB)提供了一种非生物的替代品，提高了生物安全性，加速了其通过监管机构的批准应用。本文重点介绍并讨论了近些年利用TXTL对CRISPR系统的表征及在生物传感器中的应用，希望能更好地推动CRISPR技术和TXTL在生物传感器方面的发展。

Abstract:

Cell-free transcription and translation (TXTL) system is a cell extract-based system for rapid in vitro 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.