Researcher(s)
- Jasmine Grimsley, Chemical Engineering, University of Delaware
Faculty Mentor(s)
- Kevin Solomon, Chemical & Biomolecular Engineering, University of Delaware
Abstract
Gene editing technologies have accelerated the development of microbial chemical factories, enabled the production of key biotherapeutics, and been repurposed for molecular diagnostics. However, the widely used CRISPR/Cas9 system requires a protospacer-adjacent motif (PAM) to recognize target sites, limiting its ability to access many genomic regions. To address this limitation, we are developing prokaryotic Argonautes (pAgos), which are programmable nucleic acid-guided endonucleases that cleave targets without needing a PAM site. Because pAgos have no known targeting restrictions, they offer the potential for more flexible and precise gene editing. However, their broader use is limited by the lack of efficient strategies to generate guides (~20 nt single-stranded DNA oligonucleotides; ssDNA) in vivo. I address this bottleneck by leveraging T7 RNA polymerase mutants (dRNAPs)—which can synthesize ssDNA in bacterial and eukaryotic systems—to directly transcribe pAgo guides. I compared the leading dRNAP (D10E, I581F, A586V, A615T), dRNAP-1, to a rationally engineered variant, dRNAP-2 (dRNAP-1 with an additional S641A mutation, which is expected to improve specificity). I characterized ssDNA guide production for yield and sequence fidelity via electrophoretic mobility shift assays (EMSAs), nuclease treatments, mass spectrometry, and sequencing, concluding that dRNAP-1 outperformed dRNAP-2. Functional in vitro validation demonstrated that these guides effectively direct pAgo-mediated target cleavage comparable to what others have reported in literature for chemically synthesized guides. To further improve performance, I designed ssDNA-activated genetic timers in silico using riboswitches with enhanced responsiveness to DNA over RNA triggers. I then validated that these designs suppressed mCherry expression without ssDNA-triggers present, with the goal that these regulatory elements will delay pAgo expression until sufficient guide is made, thereby reducing off-target activity and improving editing specificity. Together, this work establishes an improved strategy for pAgo guide generation and lays the foundation for ssDNA‑based logic circuits, advancing the toolkit for precise, unrestricted gene editing.