RAPID: Point-of-Need Detection of COVID-19 using CRISPR-Enabled Cell-Free Synthetic Biology

COVID-19大流行凸显了SARS-CoV-2冠状病毒实验室检测的局限性，因为它不能随着数量的突然和急剧增加而扩大。实验室检测通常基于定量聚合酶链反应，需要设备、时间、专门知识和基础设施，造成严重的后勤挑战，最终导致检测不足。迫切需要能够在需要点（PON）快速部署的诊断方法，以实现对传染病的全球监测。为了满足这一需求，该项目采用了一种新的基于CRISPR的方法，称为CRISPR等温扩增（CIA），可以在环境温度下实现SARC-CoV-2病毒的一锅诊断扩增和检测。该方案建立在无细胞合成生物学和生物传感的创新基础上，能够快速，低成本和PON检测化学污染物和病毒病原体。正在开发的平台技术可以用于多个目的，从快速筛选技术到告知分类和隔离策略，再到用于前线的临床测试。这项技术也有可能最终作为一种家庭测试，帮助告知哪些亚群和经济部门可以在大流行结束时安全地恢复生产力。此外，该测试具有快速重新编程以检测新出现的病原体的灵活性。这项工作是与商业伙伴Stemloop公司密切合作进行的，该项目采用了一种新的CRISPR等温扩增（CIA）方法，可以在病毒检测中实现SARS-CoV-2基因组的一锅扩增和检测。CIA使用编程的Cas 13系统识别SARS-CoV-2 RNA，触发核酸扩增，并在扩增发生的同一反应中同时产生可检测的输出。每个过程的计算模型与实验系统一起开发，并用于指导优化以实现阿摩尔灵敏度。 该方法的独特之处在于不需要DNA引物。放大和检测过程的设计需要不到一个小时，成本不到一美元，每个测试的制造。这个奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

The COVID-19 pandemic highlights a limitation of laboratory-based testing for the SARS-CoV-2 coronavirus in that it does not scale with a sudden and dramatic increase in volume. Laboratory tests, often based on quantitative polymerase chain reaction (qPCR), require equipment, time, expertise and infrastructure, resulting in severe logistical challenges and ultimately resulting in inadequate testing. There is a critical need for diagnostics that can be rapidly deployed at the point-of-need (PON) to enable global surveillance of infectious diseases. Towards addressing this need, this project incorporates a new CRISPR-based approach called CRISPR Isothermal Amplification (CIA) that enables one-pot diagnostic amplification and detection of the SARC-CoV-2 virus at ambient temperatures. This scheme builds off innovations in cell-free synthetic biology and biosensing that enable rapid, low-cost, and PON detection of chemical contaminants and viral pathogens. The platform technology under development could serve several purposes from a quick-screen technology to inform triage and isolation strategies, to a clinical test used on the front lines. There is also the potential for the technology to eventually serve as an at-home test to help inform which subpopulations and sectors of the economy could safely return to productivity towards the end of the pandemic. In addition, the test has the flexibility to be rapidly reprogrammed to detect new emerging pathogens. This work is pursued in close collaboration with a commercial partner, Stemloop, Inc., in order to quickly transition the technology for manufacturing, deployment, and distribution.This project employs a new CRISPR Isothermal Amplification (CIA) approach to enable a one-pot amplification and detection of the SARS-CoV-2 genome in an assay for the virus. CIA uses programmed Cas 13 systems to recognize SARS-CoV-2 RNA, trigger nucleic acid amplification and create a detectable output simultaneously in the same reaction where amplification occurs. Computational models of each process are developed alongside the experimental systems and used to guide optimizations to achieve attomolar sensitivity. The method is unique in that no DNA primers are required. The amplification and detection processes are designed to take less than one hour and cost less than one dollar per test to manufacture.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.