dual fluorescent expression with dCas9-KRAB-TagBFP any issues?

[GenomeJockey]

I'm trying to knock down an miRNA using dCas9-KRAB-TagBFP. I'm considering co-transfecting a fluorescent protein that's always on to check if the sgRNA is getting delivered properly. Has anyone tried using two fluorescent proteins in these cells? I'm concerned it might cause stress or affect cell health. I'd love any tips or protocols you can shaer

 

[ClintonClay2]

Using two fluorescent proteins can work but might stress cells if expression is high, you can try low-expression promoters or separate delivery vectors and also validate with controls to monitor cell health closely.

 

[Caitlyn P]

Yeah, running two fluoros is doable but shouldn't overlap too much in emission TagBFP is pretty blue, so something like mCherry or GFP should work fine alongside it. You could also use a separate plasmid with a strong constitutive promoter (like EF1α or PGK) driving your second FP just to track sgRNA delivery.