Hi,
I'm trying to generate p53/p16 double knockout cell line. I managed to knockout p53 using Crispr. Now, I can't manage to knockdown/out p16. I have tried so far 5 different Crispr targeting different areas. I have also tried two shRNAs, one targeting coding sequence and another targeting coding sequence and 3'UTR. Till now, I have no luck? Any thoughts?
Thanks,
Abdalla
Thanks for your answer, I measured protein level, used pX459-puro/ electroporation for transfection.
Yes, sometimes the NHEJ will not knockout the protein because the gene is still in frame. It happens 1/3 of the time, quite common. And sometimes, the NHEJ does not even result in indels.
I suggest you use a genetic assay like sequencing or surveyor to see if the gene is still in frame. Alternatively, you can knock in a GFP cassette using HDR. If the GFP is knocked in, then the p16 will most definately be disrupted. You can easily select the KO cells using GFP.
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