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.

https://media.addgene.org/cms/files/Zhang_lab_LentiCRISPR_library_protocol.pdf

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?


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Fuchsia Kelpiealmost 2 years ago

Please see the screening paper (Joung Nature Protocols 2017) for the experimental details.

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