CAREER: Characterization and Development of Prokaryotic Argonautes for Synthetic Biology

该奖项全部或部分由《2021年美国救援计划法案》（公法117-2）资助。源自细菌DNA干扰或“免疫”系统的基础技术广泛应用于现代生物技术。这些系统具有编辑基因和为各种目的重新编程生物体的能力，包括生产胰岛素和抗体疗法等重要药物，创造高产和有营养的作物，从可再生资源中产生化学品和材料，甚至治疗某些遗传疾病。原核Argonaute蛋白（pAgos）是一个未被充分研究的系统，具有在更广泛的位点编辑基因的潜力，并最终有可能扩大这些基础技术的范围。本项目旨在确定pAgo功能和性能所需的组件，并加速生物技术应用的pAgo的开发。这种新的生物技术工具的潜在应用范围很广，包括农业、可持续性、医学和诊断。这项研究的内容将被纳入本科和高中课程，并将为下一代生物技术专业人员提供研究经验。美国救援计划为该项目提供的资金将支持一名处于职业生涯关键阶段的调查人员。pAgos构成了一组不同的可编程内切酶，这些内切酶并不依赖于邻近的原间隔基序进行有效的切割。本项目的总体目标是识别和控制介导pAgo DNA干扰的因素，以加速pAgo生物技术的发展。核心假设是，pAgos依赖于保守的原核辅助蛋白来发挥功能。因此，高通量的体内功能筛选将被用于从测序基因组和土壤宏基因组中快速表征pAgo候选物。这种功能活性将与基因组证据相结合，以识别预测pAgo底物偏好和活性的特征和假定的辅助蛋白。与此同时，相互作用的辅助蛋白将被分离出来并评估其在功能中的作用。这些实验研究将为pAgo活性的机制提供新的见解，并改进现有数据库中pAgo相关基因的注释。这些研究的发现将用于真核生物基于pag的基因编辑的概念验证。本研究将通过基于发现的本科实验课程，为有前途的pAgo候选人挖掘土壤宏基因组，并吸引高中和本科研究人员，作为劳动力发展的焦点。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117-2).Foundational technologies derived from bacterial DNA interference or ‘immune’ systems are widely used in modern biotechnology. These systems have the ability to edit genes and to reprogram organisms for a variety of purposes, including production of vital medicines such as insulin and antibody therapies, creating high-yield and nutritious crops, generating chemicals and materials from renewable resources, and even treating certain genetic disorders. Prokaryotic Argonaute proteins (pAgos) are an understudied system with the potential to edit genes at a wider range of sites, and ultimately the potential to broaden the range of these foundational technologies. This project aims to identify the components needed for pAgo function and performance, and to accelerate the development of pAgos for biotechnology applications. The potential applications for this new biotechnology tool are broad and include agriculture, sustainability, medicine, and diagnostics. Elements of this research will be incorporated into undergraduate and high school curricula and will provide research experiences for the next generation of biotechnology professionals. American Rescue Plan funding for this project will support an investigator at a critical stage in his career.pAgos constitute a diverse group of programmable endonucleases that are not known to rely on a protospacer adjacent motif for efficient cleavage. The overarching goal of this project is to identify and control the factors that mediate DNA interference by pAgos to accelerate pAgo development for biotechnology. The central hypothesis is that pAgos rely on conserved prokaryotic accessory proteins for function. Thus, high-throughput in vivo functional screens will be leveraged to rapidly characterize pAgo candidates from sequenced genomes and soil metagenomes. This functional activity will be integrated with genomic evidence to identify signatures and putative accessory proteins that predict pAgo substrate preferences and activity. In parallel, interacting accessory proteins will be isolated and evaluated for their role in function. These experimental studies will provide new insight into the mechanisms of pAgo activity and improve annotation of pAgo-related genes in existing databases. Findings from these studies will be used to develop proof of concept pAgo-based gene editing for eukaryotes. This research will serve as a focal point for workforce development through discovery-based undergraduate lab curricula that mines soil metagenomes for promising pAgo candidates, and engage high school and undergraduate researchers.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.