• Austin Futty, Applied Molecular Biology & Biotechnology, University of Delaware

• Kevin Solomon, Chemical and Biomedical Engineering, University of Delaware

Prokaryotic Argonaute purification and characterization for evaluating utility in novel synthetic biology toolkit creation

    Gene editing forms an integral part of synthetic biotechnology and has become important to the food industry, healthcare, and other areas. These techniques rely upon endonucleases (proteins that cleave DNA) to edit target sites. CRISPR/Cas9 is the gold standard for gene editing due to its targeting flexibility compared to other endonucleases. However, Cas9 requires a short specific DNA sequence, called a protospacer adjacent motif (PAM), in order to make edits, restricting its use. Prokaryotic Argonautes (pAgos) are a diverse family of endonucleases that are easily reprogrammed, much like Cas9, making them viable alternatives. Importantly, pAgos have no targeting requirements, including PAM sites, expanding the scope of gene editing.

    pAgos have only been demonstrated to facilitate gene editing in bacteria and have not been done successfully in eukaryotes. Therefore, we need a strategy to characterize pAgo strains expressed in diverse hosts. Specifically, I developed a pipeline to purify His-tagged Clostridium butyricum Argonautes (cbAgo) in order to characterize them in vitro. Once purified, activity was screened against single and double-stranded targets. Activity was observed in cbAgo expressed from both E. coli and S. cerevisiae, model prokaryotic and eukaryotic species respectively. Preliminary results suggest similar activity levels between the two species. Additionally, S. cerevisiae expressed cbAgo were active across the range of GC content tested (13-43%). Although effective, traditional activity assays are cumbersome as each sample must be run on an agarose gel for analysis. A high-throughput version will simplify and expedite characterization of novel and engineered pAgos. Preliminary results suggest that pAgo activity can be monitored in real-time in the presence of ethidium bromide. In conjunction with these findings, the creation of this assay will allow further exploration of the capabilities of pAgos.