Poor expression efficiency of AAV-Mecp-spCas9 and AAV-rsv-spCas9?

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.


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Ruby Wyrmabout 4 years ago

do you know whether the tMecp2 promoter is expressed in the cell line you want to target?

More recently it has been published another SpCas9, this time expressed under the control of the short EF1a promoter. They used this Cas9 to do in-vivo knockin in the adult mouse brain neurons and it works great. We got the plasmid from Addgene and were able to reproduce the data.

Probably your system is not working because the Cas9 is not expressed?

Green Bogreabout 4 years ago

Thanks for answering.

No, I don't know if the MECP promoter expresses Cas9, didnt do the immuno in neurons yet. I would like to know how you proved Cas9 expression. Immuno, western blot, RNA expression?

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