Discovery of novel nucleases for genome editing applications

Project

Discovery of novel nucleases for genome editing applications

Discovery and characterization of novel prokaryotic Argonaute proteins with genome editing capabilities and protein engineering of characterized ones to transform them into genome editing tools.

Context

The journey to look for naturally occurring and synthetic nucleases with genome editing capabilities is not new. The pursuit of proteins that could generate double-stranded breaks has been going on for a long time, but only in the last ten years has the practical application of these nucleases become a reality.

Different approaches have been taken, like the use of meganucleases, Zinc Finger Nucleases (ZFNs), Transcription activator-like effector nucleases (TALENs), and most famously, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated proteins (Cas), forming the CRISPR/Cas system.

All the different approaches have their benefits and their drawbacks, but none of them can be considered the golden fleece. For this reason, there is a constant search for novel nucleases that could, if not become the golden standard of genome editing, at least complement the always growing toolkit for genome engineering.

Argonaute proteins were first discovered in eukaryotes, being directly involved in the RNA interference pathway (RNAi), in which Argonaute is the effector protein of the RNA-Induced Silencing Complex (RISC). The RNAi pathway is a biological process in which RNA molecules inhibit gene expression or translation by neutralizing target RNA molecules. This pathway acts both as a regulatory mechanism for endogenous gene expression, and as a defence mechanism against viruses and other genetic material.

Even though the RNAi pathway is exclusive to eukaryotes, proteins homologous to eukaryotic Argonautes were discovered in bacteria and archaea. These prokaryotic Argonautes (pAgos) have a higher structural diversity than their eukaryotic counterparts, a diversity that allows them to use DNA or/and RNA as guides, and target DNA or/and RNA, depending on the specific Argonaute. A group of pAgos, those that used DNA guides to cleave DNA targets, quickly stood out as possible tools for genome editing applications.

Aim

The project aims at the discovery and characterization of novel pAgos with the potential to be used as genome editing tools, as well as the protein engineering of pAgos that show promising characteristics but fall flat of being candidates due to specific structural drawbacks. In addition, the project aims to understand the biological role of these pAgos as well as to unravel their molecular mechanism of action, two bits of knowledge that will allow us to work with them in a more effective manner.

Techniques

The project will consist of two approaches:

  1. The systematic characterization of promising novel pAgos, which involves molecular biology techniques such as heterologous protein expression, protein purification, in vitro activity assays, etc.
  2. The protein engineering of characterized pAgos, which involves molecular biology techniques, but with a strong focus on bioinformatics and protein biochemistry.

Contact

Thesis projects are available for enthusiastic BSc and MSc students. If you have any questions regarding the projects, feel free to contact me.