I have some trouble with AAV-CRISPR/Cas9. I co-transduced prairie vole primary neuronal cultures with AAV-sgRNA against the oxytocin receptor or LacZ and AAV-MECP-Cas9 or AAV-RSV-Cas9. In both cases I dont see any editing after a T7E1 assay. Of course, there can be 1000s of reasons, but the sgRNAs are validated (with a PCR based-method and recombinant Cas9), so first I want to make sure that Cas9 is properly expressed. I did immuno's on my primary cultures for with anti-Cas9 and anti-HA antibodies, but no result.
To make things easier I transduced HEK cells with my AAV-RSV-Cas9 vectors and did an immuno to visualize the HA-tag, but all that I see is background fluorescence. I have been checking some papers and most people seem to do western blots to validate Cas9 expression. But the bands are not that intense, which is why I start thinking Cas9 is not highly expressed by the MECP or RSV promoters.
What I just like to know if more people ran into this. I know I am working with prairie vole, which complicates things a bit, but I am thinking if I should order other pAAV-spCas9 vectors (with cmv or minicmv promoters) just to try that. Or maybe not use immuno's, but western blot and/or RNA expression to probe cas9 expression.
Any thoughts on this are highly appreciated.