Researcher(s)
- Jadira Aurora Fuentes Bautista, Chemical Engineering, University of Delaware
Faculty Mentor(s)
- Kevin Solomon, Chemical and Biomolecular Engineering, University of Delaware
Abstract
Identifying essential native protein Interactors of Clostridium butyricum Argonaute
Jadira Aurora Fuentes Bautista 1, Brett Graver2, Rob Barlow1, Kevin Solomon1
1Department of Chemical & Biomolecular Engineering, University of Delaware
2Department of Biological Sciences, University of Delaware
Genetic engineering is a prominent biotechnology utilized for altering genetic material for various applications in industry and research. CRISPR is currently the preferred genome editing technology as it is considered to be cheap, accurate and highly efficient. However, CRISPR requires a protospacer adjacent motif (PAM) site for proper DNA recognition and function, which may lead to issues in certain applications. Prokaryotic Argonautes (pAgos) are a promising alternative to CRISPR/Cas systems as they can be easily programmable and have no known targeting requirements (i.e. PAM site) in vivo. A handful of pAgos have been studied in vitro with some displaying function in recombinant prokaryotic systems. However, pAgo function in eukaryotic systems has yet to be demonstrated. In vitro, pAgos have enhanced function when supplemented with DNA-modifying accessory proteins, such as helicases. pAgos lack helicase activity and may require accessory proteins for function in vivo. Supplementation of accessory proteins may be key for function in eukaryotes. To achieve this, UltraID biotinylation is being used to identify protein interactors of Clostridium butyricum pAgo (CbAgo) in prokaryotic and eukaryotic hosts. Identifying CbAgo’s protein interactome in its native host is vital to correctly determine missing accessory proteins in heterologous hosts. I am cloning a CbAgo-UltraID construct in a C. butyricum shuttle vector to facilitate these studies. Successful completion of this project will identify critical components of pAgos that are needed for function, potentially enabling further development of this novel and potentially powerful gene editing platform.