I have three plasmids with identical Cas9. EGFP is a selectable marker in all the plasmids. The plasmids are identical in all the aspects except for one; each one has a different guide RNA cloned for a single gene. I plan to co-transfect all three into one cell type and look for down-regulation. My question is how can I check that the cell has all three plasmids when the selectable marker is identical? Also, is it safe to add three Cas9 molecules into one cell?
By PCR made on genomic DNA with specific primers, if plasmids are integrative ones and the sequencing of fragments. By plasmid rescue (isolation of extra-chromosomal DNA and transformation of bacteria with residual DNA obtained) if plasmids a replicative ones and then sequencing of plasmids.
Hi Edouard, Thanks for your answer. However, I am going to use mammalian cells for transfection and the plasmids are non-integrative. Are you aware if we can use the same approach with mammalian cells as well?
What I said previously was for mammalian cells. If I am correct when transfecting different plasmids you have a gaussian curve : ie very few cells will have only one vector, most of them will have 2 or 3 vectors and other very few cells will have no vectors (depending the effectiveness of your transfection reagent used).
Applying antibiotic selection during transient expression on a few days might help cells in keeping plasmids.
Another possibility will be to have the 3 genes included in the same vector ?
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