CAREER: Characterization and Development of Prokaryotic Argonautes for Synthetic Biology

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.