Hi guys: I have been doing CRISPR knock-in and knock-out and one seemingly impossible scenario keeps happening.
In the CRISPR knock-in attempt I tried to add a tag on a gene of interest, so I transfected the plasmid with gRNA and cas9 and selected by puromycin. After that I did some quick check on the pool by PCR and Western, which were both positive. I could see the protein of interest with my tag being expressed by western.
So I moved on to start sparse plating and picked single colonies into 96-well plates. I genotyped the colonies by PCR and found some positive cells. I transferred them to 24-well plates and then 6-well plates as they grew. However, once I transferred them, they reverted to WT. I no longer saw the band by PCR, and no longer saw the tag-protein in western. When I asked other people for advice most of them suggested that the single-colony was not clonal and has WT cell contamination, and the edited cells had a disadvantage that was selected against. However I found this highly unlikely because the cells apparently existed in the pool (which must have been with a much lower percentage (say 1%) than the clones I picked, because this CRISPR itself was very inefficient). I was also able to pass down the pool cells for several generation, and still saw positive cells there by PCR. If it's outcompeted by WT cells, how come it still existed in the pool?
This happened again when I tried to do CRISPR knock-out on the same gene. For some clones in the 96-well plate, I saw indels by PCR. However they reverted to complete WT after I transferred them to larger wells.
I feel there must be something really wrong going on. I would appreciate any suggestion and advice. Thank you very much.
I agree with your colleagues - you have mixed samples of cells.
Remember, PCR is extremely sensitive and will pick up the rare cells that have your construct.
Katie A Burnette Thank you very much for your response.
But even if I had mixed samples of cells in the clones, I still don't understand how the positive cells got outcompeted. If the positive cells were selected against, why could they exist in the pool, where the majority of cells was WT?
Wildtype cells almost always will outcompete other cell types (except in the case where only the mutated cells have an antibiotic resistance marker or similar). You purposefully chose colonies that had some of your transformed cells based on your Western blot. Try streaking out to single cell colonies and/or add a selectable marker.
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