Creating knock-outs of Celiac Disease associated loci using CRISPR/Cas9

Roeland Broekema
MSc in Biomedical Sciences, University of Groningen
Internship: April – June 2016
Supervisor: Iris Jonkers

Summary

CRISPR/Cas9 is a new and promising gene editing technology. Unfortunately, it still suffers from various ‘teething’ issues. The most important one being the off-target effects it introduces during gene editing. Simply put, CRISPR/Cas9 consists of two components: the Cas9 Nuclease which cuts the genome, and the sgRNA which can be modified to target a specific location within the genome for the Cas9 Nuclease to cut. Both of these components can be stably transfected into cells of interest by means of viral transduction (i.e. permanently incorporated into the genome of the cell) to create knock-outs of a specific genomic location of interest. This results in continuous Cas9 activity at the target location as defined by your sgRNA, but more importantly, it also results in continuous off-target effects. Within a few days or weeks the cells will stop growing due to the damage caused by these off-target effects, stopping you from properly test the genomic location you actually meant to target.

The goal of my project was to avoid this continuous Cas9 activity by setting up a transient transfection method (i.e. temporarily introducing both Cas9 and the sgRNA to the cell) for the specific cell line we wanted to apply this to. Two different methods were tested to achieve transient transfection: the use of liposomes, and electroporation (nucleofection). RNA or DNA introduced into cells with these methods will not be incorporated into the genome but will instead be degraded or diffused out of the cell within a few days after transfection. The cationic properties of liposomes allows them to encapsulate the RNA or DNA plasmid you wish to introduce into a cell, followed by diffusion into the cell. Electroporation applies a very short electric pulse to induce small punctures in the cell wall, which allow RNA or DNA to enter the cells.

I tested three different types of liposomes and one electroporation method with varying degrees of success.

My experience

When I started this internship in the Department of Genetics, I had already finished my Master’s degree but wasn’t sure if I wanted start working for a commercial company to immediately, or if I wanted to first expand my in-depth genetics knowledge by doing a PhD. My experience at this department made it easy for me to choose to do my PhD because of the opportunities and exciting research that is being performed here. The people in this department all strive for the very best research and put in a lot of work to achieve this. However, at the same time, everyone knows how to have fun, keeping the general atmosphere relaxed. Research is much more exciting when you are working with new and exciting techniques, and even more so when you are working in a very stimulating and ambitious environment as is most definitely the case for this department!