Are you using primary derived cells or an immortalised cell line Ifeanyi?

Primary derived cells will increasingly become quiescent or senescent after regular passaging, which from my experience has a negative effect on siRNA-mediated knockdown - would suspect it may be the same for CRISPR-Cas9.

If it's a cell line, gene expression changes occur with increasing passage, which could affect editing efficiency.

How does the proliferation of your cells at present compare to their proliferation when the CRISPR was successful? Do you see any signs of toxicity post-transfection (i.e. dead cells floating under the microscope or stained blue by Trypan Blue)?

And do you have any vials of cells at the passage number (or even better lower) where CRISPR was successful, so that you could transfect these at the same time as your high passage cells to test your hypothesis?

Let me know your progress with this.

Good luck.

John Henderson

While you have a point that cells do been senescent with time, however the failure of gene-editing is in a set of my experimental gRNAs, while another set (my positive control gRNA) works just fine with these 'old' cells.

Also I have tried to transfect an earlier passage of cells which was unsuccessful aswell with my experimental gRNA (cells were frozen after initial purchase in 2018, not sure of the actual passage number but must've been lower or similar to the cells used when gene-editing did worked).

I do see a few 'floaters' after transfection but these are equally present in the positve control gRNA transfected wells. I'll have a go at changing the media a few hours after transfection to see if that makes a difference.

Cheers for the input, still open to suggestions and I'll keep you posted on any progress!.