Engineering Prokaryotic Argonautes for Transcriptional Regulation

• Ashley Kalan, Chemical Engineering, University of Delaware

• Kevin Solomon, Chemical & Biomolecular Engineering, University of Delaware

Engineering Prokaryotic Argonautes for Transcriptional Regulation

Ashley Kalan¹, Brett Graver², Kevin Solomon¹

¹Department of Chemical & Biomolecular Engineering, University of Delaware

²Department of Biological Sciences, University of Delaware

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. Transcriptional regulators bind DNA and recruit or disrupt transcriptional machinery to increase (activate) or suppress (interfere with) gene expression. A common strategy uses the catalytically dead version of the CRISPR Cas9 protein, which can be programmed to regulate a selected gene. However, the effectiveness of 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. Prokaryotic Argonautes (pAgos) are a promising alternative to the CRISPR Cas system because these targetable endonucleases have no known recognition restrictions in vivo, allowing for optimal gene targeting. Here, my experiment suggests that the catalytically dead pAgo from Clostridium butyricum (dCbAgo) transcriptionally interferes with essential gene expression in a recombinant Escherichia coli system. To measure genetic interference, dCbAgo was expressed to target essential antibiotic resistance genes on a target plasmid or in the cell’s genome. dCbAgo mediated interference of these genes led to lower cell survival rates in the presence of antibiotics. A fluorescent reporter assay was also developed to study transcriptional activation and interference. To capture the dynamics of gene regulation and validate performance, I cloned a ssrA degradation tag on the c-terminus of the fluorescent reporter protein, mNeonGreen, which has been validat . Continuing to engineer pAgos as a tool for transcriptional regulation will provide the synthetic biology field with a more flexible, alternative biotechnology to CRISPR interference/activation.