Researcher(s)
- Timothy Merrill, Biological Sciences, University of Delaware
Faculty Mentor(s)
- Timothy Merrill, Department of Chemical and Biological Engineering, University of Delaware
Abstract
Gene editing has rapidly advanced over the last decade with the adoption of CRISPR/Cas9, an RNA-guided endonuclease known for its efficiency and ease of use. Unlike earlier tools that required extensive protein engineering to reprogram, Cas9 enables consistent DNA targeting simply by altering the single-guide RNA (sgRNA) spacer sequence. However, Cas9 has notable limitations: its large size complicates delivery in medical contexts, it is entangled in intellectual property restrictions, and it requires a protospacer adjacent motif (PAM) at the target site, an element not always present at optimal positions. This PAM dependence restricts Cas9’s range of editable sequences, creating demand for a more universally targetable editing platform.
Prokaryotic Argonautes (pAgos) are a promising candidate for next generation gene editors. Like Cas9, they use nucleic acid guides, making them easy to reprogram, but pAgos do not rely on PAM sites, allowing them to target any sequence. Despite their promise, challenges remain, including non-specific nuclease activity (“chopping”), poor targeting of double-stranded DNA (dsDNA) unless aided by accessory proteins, and low activity at mesophilic temperatures (approximately 37°C).
To address these hurdles, we selected four of the most widely used pAgos in the field for engineering through directed evolution. This process involves generating libraries of random mutants through error-prone PCR, selecting for “better” versions (i.e., those with improved activity against dsDNA), and repeating the cycle, mimicking natural selection at an accelerated pace. Selection will be carried out using an assay in which pAgos edit E. coli to knock in an antibiotic resistance gene; only cells with functional pAgos will survive. Our goal is to evolve pAgos with enhanced activity and reduced off-target effects, advancing their potential for safe, efficient gene editing without the constraints of PAM site dependence.