DNA is edited but protein still remains, is this a problem?

A while back I performed a genome-wide CRISPR dropout drug screen in a leukemia cell line and found some pretty cool hits. I then followed up the screen with some validation experiments where I performed a reporter (RFP)-based competition assay that was assessed via flow cytometry. In my screen I used the Brunello library with 4 sgRNAs/gene. I selected two from the library and cloned them individually into the SGL40C.EF879S.tRFP657 lentiviral vector. I transduced my Cas9-exressing cells at ~60-70% with a control sgRNA, sgRNA1 & sgRNA2, treated the cells with drugs and monitored the fluorescence of the cells over time via flow. I was able to independently validate that my sgRNAs targeting my gene indeed sensitized my cells to the drug.

Following this experiment, I sorted all the RFP+ cells from my control populations and did some downstream analyses. With TIDE analysis, I had ~80% editing efficiency with both sgRNAs. sgRNA1 revealed that most of my edits had a 10bp deletion whereas sgRNA2 revealed that most of my edits had a 1bp insertion (which was very cool to see given that in my validation experiment sgRNA1 performed better than sgRNA2). Either way, it looked like a frameshift should occur. However, when I did a western blot using these cells, the protein level in my edited cells looked exactly the same as my control population (similar molecular weight as well, the antibody was detecting epitopes upstream of my cut sitel). I also did a qPCR on all of these populations and the transcript level was nearly identical.

I’m a bit puzzled as I was expecting a drop in protein level at the very least. In essence I’ve confirmed that my DNA is edited but the protein remains. My protein has 3 exons and my sgRNAs target the beginning portion of the 3rd exon. From the literature the dominant isoform has all 3 exons translated whereas the 2nd isoform only encodes exons 1 & 3. Given that my sgRNA is targeting the 3rd exon (common to both isoforms), I kind of ruled out that another isoform is dominating? Is it possible that the protein that’s remaining is simply mutated in the edited populations, is this common? And if it's mutated I guess I'd have to show whether the remaining protein has a loss of function, but I'm unsure how to go about that as my protein isn't well studied at all. I guess my main concern is, is this result a major concern? I’d like to move on to doing more downstream experiments to study more mechanism but I’m unsure/a bit worried how important it is to have the protein gone. Any insight would be very much appreciated as I’m not too well versed in the CRISPR world & neither is my group.

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Green Gnomealmost 2 years ago

I think you found the protein intact because of the iilegitilate translation of the protein. Sometimes when there is a frameshift mutation in the DNA, the cell skips these mutation and reinitiate the translation.

But I am not sure whether this affects the protein function of not. If I were you, I will plan to delete the whole protein using 2 gRNA, if possible.

Hope this helps!

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