So I'm doing CRISPR this week. Starting with 30 million stem cells, I worked my way down to about 500,000 that survived electroporation, 2% of which actually showed a marker of Cas9 expression. Of those, maybe 10% will survive this week and grow from single cells back into full colonies without losing their pluripotency. Of those, maybe 10% will actually have a change that I want (destroying gene function). I believe it'd be an order of magnitude lower if I actually wanted to actually wanted to make a mutation in a more targeted way. So 0.0003% of my starting cells will actually be gene edited and usable afterwards. I personally only need about 10 in total to make it through the whole process, but it's hard to look at those numbers and feel we're a longgg way from designer babies.
Yeah, the technique is still in its infancy, and targeting is still problematic. But maybe you are the person to innovate a "rifle scope" for CRISPR? Who knows? Accuracy will come. Science will refine the methods and techniques. And you are a part of that. YOU ARE SO COOL!!!! I'm jealous.
Doubtful, I'm just using it to make separate cell lines, I have zero plans to optimize anything if I can get my 10 cells that look alright by the end of the year. But thanks, it's nice to hear encouragement.But maybe you are the person to innovate a "rifle scope" for CRISPR? Who knows?
"Nucleofector – Amaxa" and "Human Stem Cell Nucleofector Kit 1 – Lonza", I may need to play around with it though, as the protocol appears to have not worked very well (it was ~6M across 4 cuvettes, which may not be the optimal cell:cuvette ratio?...)
I used Mirus solution. It's been a while. That does seem like a lot of cells. For hMSCs, I'd do more like 500k per cuvette if I remember correctly, but they are big. Also, it was key that the volume was exact. I think it was 200ul. I want to say that I used the A-27 program, but I don't know if that is conserved.