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:
This dot plot showing the side scatter (SSC) and the forward scatter (FSC) of 10,000 treated cells. SSC and FSC provide an indication of the size and texture of the cells allowing live and dead cells to be differentiated. The live population has been outlined, and selected for further analysis.
This scatter plot shows the intensity of PE and Alexa Fluor 488 fluorescence (live cells only). PE intensity indicates the level of auto-fluorescence, because this stain wasn’t actually used, and helps separate the negative control population (blue) from the positive cells (in the black triangle) in the treated sample (red).
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.
Hi Elizabeth,
Have you taken a look as the PrimeEditing paper in Nature last month? If it works as well as they say it does it should be a lot easier to put small mutations(e.g. Point mutations) into your cells with much lower off target effects.(the enzyme doesn’t cleave the DNA you just right the change into the genome).
https://www.nature.com/articles/s41586-019-1711-4
Yes, I’ve seen that paper and it does look like a wonderful technique! We just wanted to stick with a technique that works with electroporation of RNPs because it seems deliver the components at much higher efficiency than any plasmid transfection we’ve done.
That’s great, can just elektroporotion also help to diminish the tumortissue like with prostatecancer?
And is there also a possiblity to use prions and change them. I mean if with Creutzfeldt-Jakob braincells turn into mush, why couldn’t we use prions to target a specific cancercell?
Interesting ! I’d never heard about Irreversible Electroporation for prostate cancer. I’ve not heard IRE being used in brain tumours, but there were a handful of abstracts covering “Tumour Treating Fields” for brain tumours at the AACR Annual Meeting this year (here’s one of them: https://cancerres.aacrjournals.org/content/79/13_Supplement/CT008).
I don’t know a lot about prions, but I don’t think they would specifically kill cancer cells over normal cells, and can’t immediately see anything in the literature about anyone else looking at this as a therapeutic option.