As soon as possible I’m hoping to use CRISPR/Cas9 genetic editing to make sets of DIPG patient cells that differ only in one gene (either with/without the whole gene or with/without mutations of interest). This will allow me to make far more accurate comparisons of behaviour between the cells in relation to specific genes or mutations.
There are many different ways to get the CRISPR/Cas9 components into the cell, but I’ve decided to send it in as the Cas9 protein and sgRNA itself (usually these are called ribonucleoprotein complexes or RNPs). This way the CRISPR/Cas9 system is ready to go as soon as it enters the cell, it won’t be as stressful to the cells as they won’t have to produce bucketloads of Cas9, and it should be more accurate because with less Cas9 in the cell for a shorter period of time there’ll be fewer chances for an error to occur. On the other hand, I have no way to include an easy to spot or select marker to find the cells in which it has worked in the end… so it’s a bit swings-and-roundabouts.
My first step is to optimise the specific method by which I deliver the components to the cell, because each cell type requires a different set of parameters. I’ve chosen to use electroporation, a technique where a strong electrical pulse breaks holes in the outer membrane of the cell, and lets the components just float in. Because my system includes no markers for the optimisation I’m having to replace Cas9 with a fluorescent antibody (called AlexaFluor488), an technique I’ve seen other people using. I can then use a technique called flow cytometry to measure the fluorescence of every single cell to check if the antibody was able to enter the cell.
The results can be visualised as plots like this:
From this I can see that 85% of the cells are alive after treatment, and 99% of those live cells have taken up antibody. There was a decent chance this wouldn’t work at all, but instead it was so effective that the postdoc I was working with said Wow!
You can read full details of the cells used, the method of electroporation, and the analysis completed in this Zenodo post.