Hello everybody,
I would need help because I'm struggling a bit after cloning an Anti-CRISPR protein, AcrII4A. This protein is supposed to bind to the Cas9 protein and to inhibit it and I'm using it against a VPR-dCas9 in a Luc assay. I took the original plasmid from a paper, in which the AcrII4A protein is expressed under the control of a EF1alpha promoter, together with an mCherry(mCherry-P2A-AcrII4A). Moreover, at the end of the AcrII4A, a WPRE sequence is present. Since this is a lentiviral vector, other elements are present in the backbone, even though I don't think they influence protein expression. Since I needed to work with this protein only and I also needed to modify it in order to increase the efficiency, I cloned it under the control of a CMV promoter, without the mCherry(CMV-NLS-AcrII4A-SV40polyA). I tested my construct in HEK293T and CHO cells and it was working fine, repressing the Luc expression in the presence of VPR-dCas9 + gRNA. Testing it together with the original plasmid, I noticed that this one was working way better, repressing the signal more than my construct, with a fold change that was 5-10 times higher. I thought that since this protein is quite small(86 aminoacids), the presence of P2A and mCherry was stabilizing the RNA. I was also thinking that the WPRE elements was enhancing the expression. I thought about the promoter, but CMV is supposed to be strong as well, compared to EF1alpha. Another hypothesis is that the expression of the mCherry is creating burden, lowering the expression of the Luc. Do you have any explanation? Should I clone it in a different way? Thank you a lot for your help! This is the original plasmid:
https://www.addgene.org/125148/
- Badges
- Science topic
- Similar topics
- Biological Science
- Histology
- Fixatives