Engineering a Mesophilic Prokaryotic Argonaute for Transcriptional Regulation

Prokaryotic Argonautes (pAgos) are programmable endonucleases that use a small oligonucleotide guide to target a nucleotide substrate. Currently, pAgos have been deployed in diagnostics and have the potential for in vivo gene editing. Active pAgos can nick a nucleotide substrate with a catalytic tetrad (residues DEDX, where X is D, H, or N) which is responsible for its endonucleolytic cleavage. However, one potential biotechnology application of catalytically inactivated pAgos is transcriptional regulation. Transcriptional regulation is central to biomolecular engineering, enabling diverse applications such as sustainable energy production, synthesis of medicinal compounds, treatment of disease, and development of climate resilient crops. A common strategy uses the catalytically dead version of the CRISPR Cas9 protein usually fused with a transcriptional activator or repressor. This Cas fusion can be programmed to regulate a selected gene of interest; however, the effectiveness of the Cas9 system is restricted by a protospacer adjacent motif (PAM) site requirement for target recognition. Because PAM sites are not evenly distributed across genomes and are less abundant in GC-biased organisms, it is difficult to optimally target genes in certain cases. pAgos, on the other hand, are a promising alternative to the CRISPR Cas system because they have no known recognition restrictions in vivo, allowing for optimal gene targeting. Here, my work investigates the application of the pAgo from Clostridium butyricum (dCbAgo) for transcriptional regulation. To better understand the properties of each residue in the catalytic tetrad, I made point mutations in CbAgo to determine which residues alter protein activity. To further investigate the degenerate ‘X’ position in the tetrad, I made mutations with other metal coordinating residues to determine changes in activity. Assessing these CbAgo mutants, I found that non-native residues at the ‘X’ positions results in different activity levels, yet not abolished, in comparison to the wild type aspartic acid. Additionally, I determined single and double alanine point mutations within the catalytic tetrad which inactivate CbAgo. These catalytically dead mutant pAgos can be further characterized in vitro to determine binding strength and targeting restrictions. To determine the transcriptional interference capabilities of the catalytically dead version of CbAgo (D541A, D611A), I built a fluorescent reporter assay which utilizes a green fluorescence protein fused with a C-Terminal LAA degradation tag. This system allows the study of the current transcription rates of the protein by this active protein degradation turnover. Through this, I demonstrated dCbAgo mediated interference when targeted to the promoter region of the fluorescent protein gene, achieving a 30% knockdown. Future work will determine optimal targeting regions as well as targeting restrictions with the potential for implementing transcription factor fusions to dCbAgo. Continuing to engineer pAgos as a tool for transcriptional regulation will provide the synthetic biology field with a more flexible, alternative biotechnology with applications across biomolecular engineering.