dCas9-VP64 Lenti System
Apologies for what is probably a simple question. I'm essentially hoping to mentally generate a user guide for dCas9 (https://www.addgene.org/browse/article/9503/) akin to the PDF for the LentiCRISPR protocol, but for the dCas9 system.
Am I right that to target a gene for gain of function I should generate Lentivirus with the:
lenti dCAS-VP64_Blast, Retro, Gag/Pol and infect my cell of interest with these.
In parallel, subclone gRNA into lenti sgRNA(MS2)_puro backbone using the BsmBI sites and generate lentivirus.
After selecting cells with blasticidin to ensure expression of dCas9-VP64, then treat these cells with gRNA lentivirus and select with puromycin.
Are there any other plasmids that I need? Are there any other steps that I'm missing?
Please see the screening paper (Joung Nature Protocols 2017) for the experimental details.