Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts.
Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts.
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DOI：
10.3389/fbioe.2021.727584
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发表时间：
2021
影响因子：
5.7
通讯作者：
Federici F
中科院分区：
文献类型：
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作者：
Arce A;Guzman Chavez F;Gandini C;Puig J;Matute T;Haseloff J;Dalchau N;Molloy J;Pardee K;Federici F
无细胞基因表达系统已成为一个有前途的平台，现场部署的生物传感和诊断。当与基于可编程支点开关的RNA传感器结合时，这些系统可用于检测任意RNA，并冷冻干燥以用于室温运输到需要的地方。然而，这些传感器主要使用重建的PURE无细胞蛋白质表达系统来实现，由于其高商业成本和冷链运输要求，这些系统难以在全球南方获得。立足点传感器的裂解物工作的初步演示的基础上，我们描述了RNA立足点开关为基础的传感器，可以在本地生产，并减少两个数量级的传感器的成本的快速原型。我们证明了这些内部细胞裂解物提供的传感器性能与商业PURE无细胞系统相当。我们使用CRISPRi策略进一步优化这些裂解物，通过敲低负责线性DNA降解的基因来增强线性DNA的稳定性。这使得能够直接使用PCR产物快速筛选新设计。作为概念验证，我们为植物病原体马铃薯Y病毒（PVY）开发了新型立足点传感器，该病毒显著降低了这种重要主食作物的产量。低成本无细胞立足点传感器在当地的实施可以在区域一级实现生物传感能力，并导致更分散的传染病全球监测模式。
Cell-free gene expression systems have emerged as a promising platform for field-deployed biosensing and diagnostics. When combined with programmable toehold switch-based RNA sensors, these systems can be used to detect arbitrary RNAs and freeze-dried for room temperature transport to the point-of-need. These sensors, however, have been mainly implemented using reconstituted PURE cell-free protein expression systems that are difficult to source in the Global South due to their high commercial cost and cold-chain shipping requirements. Based on preliminary demonstrations of toehold sensors working on lysates, we describe the fast prototyping of RNA toehold switch-based sensors that can be produced locally and reduce the cost of sensors by two orders of magnitude. We demonstrate that these in-house cell lysates provide sensor performance comparable to commercial PURE cell-free systems. We further optimize these lysates with a CRISPRi strategy to enhance the stability of linear DNAs by knocking-down genes responsible for linear DNA degradation. This enables the direct use of PCR products for fast screening of new designs. As a proof-of-concept, we develop novel toehold sensors for the plant pathogen Potato Virus Y (PVY), which dramatically reduces the yield of this important staple crop. The local implementation of low-cost cell-free toehold sensors could enable biosensing capacity at the regional level and lead to more decentralized models for global surveillance of infectious disease.
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影响因子：
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作者：
Didovyk A;Tonooka T;Tsimring L;Hasty J
通讯作者：
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