EAGER: Quantifying SARS-CoV-2 antigen-antibody binding using cell-free guide RNA display

缺乏可靠和广泛可用的SARS-CoV-2抗体检测留下了许多悬而未决的问题。例如，尽管已知SARS-CoV-2特异性抗体会在某人感染病毒后两周内出现。然而，有几个因素是未知的，包括a)产生抗体反应的病毒载量；b)SARS-CoV-2抗体持续多久；或c)保护所需的抗体量。因此，创建低成本、高精度的SARS-CoV-2抗体分析平台可以在跟踪新冠肺炎的传播、为流行病学反应提供信息以及支持诊断、治疗和疫苗的开发方面发挥关键作用。该项目将开发一种低成本的替代方案，用于快速检测SARS-CoV-2抗体结合。这些系统可以用来测量患者样本中的SARS-CoV-2抗体效价，使研究人员能够更好地模拟新冠肺炎的传播和了解其病程。这一可定制抗体检测平台的开发也将使其更容易应对未来的新流行病。此外，该平台作为一种通用研究工具具有很大的潜力，因为它可以用于检测各种抗原-抗体相互作用。更广泛的影响：拟议的系统将提供一种可扩展的、低成本的ELISA替代方法，可用于测量患者样本中的SARS-CoV-2特异性抗体效价，并促进为新冠肺炎的传播建模和了解其病因的努力。该项目的直接更广泛的影响还包括i)为博士和本科生提供了参与受使用启发的科学和工程的独特机会，ii)与不同的公众受众交流关于新冠肺炎抗体检测科学的关键思想，以及iii)关于新冠肺炎抗体检测科学的图形材料，供在面向公众的活动中使用和在社交媒体上分享，将根据Creative Commons许可免费获得。该项目将开发一个无细胞转录和翻译(TXTL)平台，用于测量SARS-CoV-2抗原-抗体结合，具有ELISA的定量精度。以及一个完全由基因编码的系统的轻松可定制性和可扩展性。最近的工作开发了一种针对大肠杆菌和TXTL的可编程CRISPR-Cas转录激活(CRISPRa)系统，该系统使用修饰的引导RNA(GRNAs)来招募转录激活剂。这里的目标是设计CRISPRa作为一个量化抗原-抗体结合的平台，方法是使CRISPRa的活性以样本中是否存在IgG抗体为条件。直接将CRISPRa活性与可见输出联系起来，这里开发的系统可以提供一种可扩展的、低成本的ELISA替代方法，允许仅使用DNA和TXTL母体混合物作为试剂来定量抗体滴度。更重要的是，由于CRISPRa固有的多路传输能力，将有可能开发新的诊断方法，仅在检测到多种抗原时才产生信号，从而降低假阳性率。智力优势：开发可扩展的方法来快速量化抗原-抗体结合是长期存在的科学和工程挑战。通过开发将抗原-抗体结合与CRISPRa导向的报告基因表达相结合的规则，该项目将创建使用DNA编程的无细胞平台来测量SARS-CoV-2抗原-抗体结合的检测方法，这些平台易于定制，并可以很容易地重新配置为护理点诊断。虽然有一些基于CRISPR的工具正被用于检测病毒核酸，但所提议的系统作为蛋白质抗体检测的传感器是相对独特的。这个奖项是由分子和细胞生物科学部门的系统和合成生物学项目利用冠状病毒援助、救济和经济安全(CARE)法案的资金颁发的。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

The lack of reliable and widely-available SARS-CoV-2 antibody testing has left many unanswered questions. For example, although it is known that SARS-CoV-2 specific antibodies appear within two weeks of someone becoming infected with the virus. However several factors are unknown, including a) the viral loads that generate antibody responses; b) how long SARS-CoV-2 antibodies last; or c) the amount of antibody needed for protection. The creation of low-cost, high-accuracy platforms for SARS-CoV-2 antibody analysis could therefore play a crucial role in tracking the spread of COVID-19, informing epidemiological responses, and supporting the development of diagnostics, treatments, and vaccines. This project will develop a low-cost alternative for rapidly measuring SARS-CoV-2 antibody binding. These systems could be used to measure SARS-CoV-2 antibody titers in patient samples and allow researchers to better model the spread and understand the disease course of COVID-19. The development of this customizable antibody detection platform will also make it easier to respond to future novel pandemics. In addition, the platform has substantial potential as a general research tool, as it could be used to detect a variety of antigen-antibody interactions. Broader impacts: The proposed system will provide a scalable, low-cost alternative to the ELISA that could be used to measure SARS-CoV-2-specific antibody titers in patient samples and facilitate efforts to model the spread and understand the etiology of COVID-19. Immediate broader impacts of the project also include i) unique opportunities for Ph.D. and undergraduate students to participate in use-inspired science and engineering, ii) engagement with diverse public audiences to communicate key ideas about the science of COVID-19 antibody testing, and iii) graphic materials about the science of COVID-19 antibody testing for use in public-facing events and sharing via social media that will be freely available under Creative Commons licenses.This project will develop a cell-free transcription and translation (TXTL) platform for measuring SARS-CoV-2 antigen-antibody binding with the quantitative precision of ELISA, and the easy customizability and scalability of a system that is entirely genetically-encoded. Recent work developed a programmable CRISPR-Cas transcriptional activation (CRISPRa) system for E. coli and TXTL that uses modified guide RNAs (gRNAs) to recruit a transcriptional activator. Here, the goal is to engineer CRISPRa as a platform for quantifying antigen-antibody binding by making CRISPRa activity conditional to the presence of IgG antibodies in a sample. It is straightforward to couple CRISPRa activity to visible outputs, and the system developed here could provide a scalable, low-cost alternative to ELISAs that permits the quantification of antibody titers using only DNA and TXTL master mix as reagents. Even more, because of the inherent multiplexing capabilities of CRISPRa, it will be possible to develop new diagnostics that produce a signal only upon detection of multiple antigens, resulting in lower false-positive rates. Intellectual merit: Developing scalable approaches to rapidly quantify antigen-antibody binding is a long-standing scientific and engineering challenge. By developing rules to couple antigen-antibody binding to CRISPRa-directed reporter gene expression, this project will create assays for measuring SARS-CoV-2 antigen-antibody binding using DNA-programmed cell-free platforms that are easy to customize and could be readily re-configured as point-of-care diagnostics. While there are a number of CRISPR-based tools that are being adapted to detect viral nucleic acids, the proposed system is comparatively unique as a sensor for protein antibody detection. This EAGER award is made by the Systems and Synthetic Biology Program in the Division of Molecular and Cellular Biosciences, using funds from the Coronavirus Aid, Relief, and Economic Security (CARES) Act.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.