I generated these RKO Cas9 stable cell line to knock out p53 using different sgRNA (one at a time). To do that, I need to induce the expression of Cas9 with Doxycyclin. I have been using 4ug/ml for 7 days. On my polyclonal population, I see a slight reduction of p53 protein using WB. However, when I check my clones, there is no knockout, only knockdown as observed in the polyclonal cells.
I was wondering if I increase the induction time and concentration of Doxy could help me generate these knockouts. I know that if Doxy is too high, it can kill my cells, but I can't think of any other solution at this time. Will my chances of getting off-target effects from Cas9 increase? Any thoughts? Thanks, guys!
Welcome to the caveats of the gene editing world.
ABC transporters in rapidly dividing cell lines, anybody?
Doxycyclin-mediated induction needs to be optimized for each cell line. based on our experience, the time of induction for 7-days is too long. One could get induction in 14 -24 h. Therefore, a time course may help. Further, the p53 protein has a short half-life. Therefore, a reduction needs to be robust under optimum KO condition.
You do not need a stable cell line with Cas9 in the first place because if the promoter is leaking then you will have problem with off-target effects of Cas9 protein. The time to induce Cas9 expression under normal CMV promoter is normally 24h but 7 days is too long and you will get off-target effects. The problem normally comes from gRNA not Cas9 expression. You have to try different gRNAs. To increase gRNA efficiency you can design three gRNAs and pool them in one transfection reaction.
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