My IPSC clones (not estbished lines) are fargile that when i transfect them with Cas9 palsmids - the cells die. I have used lipofectamine, and electroporation method for transfection. But cells dont survive.
what should i do different?
What gene are you trying to knock out. Maybe it is essential or your gRNA is cutting some off-target site which is.
Have you considered transforming them and then inducing them into an IPSC state?
its ZNF142 gene - we designed gRNAs with minimal off-target sites.
What do you mean by transforming them and then inducing them? Can you elaborate a bit..
DNA transfections can be toxic, to a degree that really depends on cell type. Have you tried different concentrations / ratios of DNA?
Otherwise to can try electroporation with Cas9/sgRNA RNPs. In other words transfect Cas9 protein complexed with the sgRNA into the cells. I haven't done this with iPSCs but I have found that it permits knockouts in cells that really don't do well with DNA transfections
https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2FMAN0017066_TrueCut_Cas9_Protein_v2_UG.pdf
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