Hello,

from my experience with hESC, generating a single K.O. line can take up to several months, notably to make sure you have a homozygous K.O. For one K.O., I would advise you to use several sgDNAs targeting several exons (preferably the first ones) to make sure the gene is really K.O. and the protein not functional in one shot.

Knocking out a second gene is already tedious work. You might consider doing it in a second round, using your first K.O. line, since the probability to cut both genes and both copies of each gene (homozygous K.O.) is really low.

From what you are proposing with using different selection and fluorescent markers simultaneously, I imagine you are asking about the feasibility of knocking all 3 genes at the same time. I think it is very unlikely, but that might also depend on the viability of the cell line.

Which cell line do you use? hESC? HEK293?

(For stem cells at least, the high mortality of cells after each step, e.g. transfection, FACS sorting, really limits the probability to get the right lineage, as most cells do not survive the experiment)