I am using 293T-BFP reporter cell. I am doing gene editing with Cas9. 293T-BFP cells have repair template plasmid and when cas9 cuts BFP site on template, BFP converts GFP by adding donor DNA.
After transfection, Although repair template has a BFP monocopy site, I observe either BFP or GFP on same cell. I don't understand why?,
Thank you for your interest !
Ramachandramouli Budida
If I understand correctly, you are observing both GFP and BFP in the same cell instead of the only GFP after Cas9 processing. How many copies of the plasmid are in your cell?. If a cell contains more copies of the plasmid with BFP, there is the possibility of cas9 cutting only some copies which leads to the expression of both colors in the same cell.
I hope this helps. Goodluck
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