CRISPR-Enhanced: Towards better genome editing nucleases by directed evolution

Background

Guided nucleases have revolutionized genome engineering. Zinc finger nucleases (ZFNs) and transcription activator -like effector nucleases (TALENs) were the first systems that allowed highly specific targeting of DNA-sequences. However, the protein engineering prior to successful DNA-targeting by ZNFs or TALENs is intensively laborious. CRISPR associated proteins have allowed for obtaining targeted DNA-modifications with unprecedent ease. However, they still do have limitations with respect to activity and specificity.

Prokaryotic Argonauts (pAgos) are another class of guided nucleases involved in bacterial innate immunity. They have shown clear capabilities of inducing DNA-cleavage under specific conditions and promise for unrestricted targeting flexibility.

Aim of the research

We aim to build a directed evolution platform to screen artificially-made Cas protein variants that will surpass their predecessors in terms of specificity and flexibility. As CRISPR proteins are well characterised, we also aim to adapt the system to search for pAgos variants that efficiently induce double strand DNA cleavage in a wider set of conditions.

Our platform is an in vitro system that includes the generation of artificial compartments through microfluidics technology, cell-free gene expression and a screening/selection system.

Techniques

Depending on the phase of the project a BSc or MSc students will have the opportunity to familiarize themselves with:

• Molecular Cloning
• Microfluidic chip design and building
• Cell free gene expression
• Flow cytometry, fluorescence activated cell sorting
• Random mutagenesis
• Design and test of screening/selection systems

Student thesis

Would you like to join this project during your thesis? Please contact: eugenios.bouzetos@wur.nl.