Bioprospecting prokaryotic Argonautes for in vivo biotechnology applications

• Kendall Massey, Chemical Engineering, Purdue University

• Kevin Solomon, Chemical Engineering, University of Delaware

Prokaryotic Argonautes (pAgos) are programmable endonucleases with potential to be an alternative to CRISPR/Cas systems. While CRISPR/Cas systems require a protospacer adjacent motif (PAM) site for targeting, pAgos have no targeting restrictions. This allows a more flexible approach to gene editing for in vivo biotechnology, as pAgos have the potential to make edits that are inaccessible to Cas systems. Despite the identification of hundreds of suspected pAgos, which span an estimated 32% of archaea and 9% of bacteria, they are understudied, with only 19 fully biochemically characterized. Therefore, it is unlikely that we have found the best candidate for in vivo biotechnology. Although pAgos are functionally diverse, an ideal pAgo would be small, use 5’ hydroxyl DNA or RNA guides, and is without intrinsic guide generation. Current approaches have been limited by their dependence on sequenced genomes; thus bioprospecting is a promising approach to discover novel genes of interest from unsequenced genomes. Bioprospecting is an established method that has been used for pharmaceuticals and identified novel Cas systems from unidentified microbes without sequenced genomes. Here, I used bioprospecting as a method to discover novel pAgos from soil samples. I designed degenerate primers with ClustalOmega and DegenPrime using 420 mesophilic pAgo sequences (identified through a bioinformatic analysis) in order to identify novel pAgo candidates. From this, I obtained five primer sets with ~60% coverage. Genomic DNA was isolated from soil samples found on University of Delaware’s campus, and genes were amplified using PCR. These suspected pAgo genes will be validated by sequencing and screened for activity using a positive selection assay. This research will allow for novel pAgo discovery without dependance on sequenced genomes and potentially find a more suitable pAgo for gene editing and biotechnology.