Hi! We used CRISPR/Cas9 for knock in of a fluorescent reporter in a cell line, in a way that our endogenously expressed target protein will be fused to RFP with a GS-linker in between.

After transfection we expanded the cells and from FACS analysis we indeed detect fluorescent cells. We sorted these RFP+ cells and cultured them again and when we FACS analyze these (RFP+ sorted) cells there is a large proportion of cells (20-30%) without fluorescence (RFP-). Via immunofluorescence we do see our target protein also present in the RFP- cell population.

Does anyone know the reason for this?

As initially only a minority of cells in our cell line express the target protein, but the majority of cells in the RFP+ as well as RFP- population do express it (as detected with immunofluorescent staining), I don't think the reason is a mistake in the sorting process as we clearly enriched for cells with the target protein. Further, as RFP and target protein are fused, I would think that the half life should be the same?

Anyways, we will generate single-cell derived clones but I am curious to know what happens there. Thank you!