Development and Optimization of a Prokaryotic Argonaute Base Editing System

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

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

In recent years, CRISPR/Cas systems (the current gold-standard for gene editing) have been leveraged in a process known as base editing, where point mutations can be introduced without the use of a double-stranded break (DSB). Base editing avoids the risk of random inserts and deletions associated with DSBs, allowing small precision edits – such as in correcting single nucleotide polymorphisms – to be made with greater reliability. However, CRISPR/Cas’s targeting mechanism relies on the presence of a protospacer adjacent motif (PAM) site nearby the target sequence, restricting CRISPR/Cas’s utility in PAM-poor regions. Prokaryotic Argonautes (pAgos) are a diverse family of programmable endonucleases with no PAM site or other targeting restrictions, providing gene editing capacity in areas currently inaccessible with existing CRISPR/Cas strategies. Despite rising interest in pAgos, their ability to perform base editing has thus far gone unreported in literature. Cas9 adenine base editors, which convert adenine to guanine, feature a base editor protein fused to a Cas9 protein via a linker sequence. To evaluate the effectiveness of pAgos in this tole, Cas9 was replaced with either CbAgo , a highly active mesophilic (~37ºC) pAgo protein, or dCbAgo, a catalytically dead variant unable to cleave. To demonstrate proof-of-concept in vivo activity, the pAgo base editors were transformed into E. coli alongside a target plasmid, supplemented with guide, and induced. Subsequent sequencing confirmed base editing, with initial editing rates approaching 7% for dCbAgo. Further experiments will be performed in order to improve the efficiency of the technique and fully characterize the system as an alternative to CRISPR/Cas base editing. This research opens the door to further applications for the use of pAgos in gene editing applications outside of areas that have historically been inaccessible by existing technologies.