Cas9 Expressing Cells Experiment

[Angelina.Mc]

I've been running an experiment targeting a specific locus using Cas9 expressing cells (stable line), and I'm running into a weird issue post transfection, a noticeable chunk of the cells just die off within 24 to 48 hours and apparently its messing with viability and downstream analysis, yes Cas9 can have some background toxicity, but this feels a bit more

I've ruled out transfection reagent toxicity already because the controls look fine, and I'm using sgRNAs that have worked in other setups. Has anyone here had similar viability issues in Cas9-stable lines or is there something I'm missing. Appreciate any help or pointers in the right direction

 

[ClintonClay2]

Yeah, Cas9 can cause stress, but sudden cell death like that could mean off-target effects or excessive DSBs, maybe check sgRNA specificity or try lower doses to ease the load

 

[DrRespectful]

The cell death youre seeing is most likely due to the sgRNA causing more damage than Cas9 alone, possibly from or on / off-target effects. Even if the sgRNAs worked before, different cell lines or high Cas9 expression can increase toxicity. Try lowering the sgRNA dose or checking for offtarget activity to improve viability