CRISPR lentiviral activation

Is it recommended to transduce all three lentiviral particles simultaneously and perform a triple antibiotics selection? Or would it be more prudent to transduce each particle sequentially and perform individual selection?

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Mauve Dragonalmost 5 years ago

Are you using the SAM activation library? It's generally better to do it step-wise (as mentioned in the Joung et. al. Nat. Protocols paper) since doing a triple antibiotic selection might potentially have toxic effects on your cells and you are adding another layer of variability to the screen. I would suggest generating stable cell lines first and then using those to perform the screen.

Hope this helps!

Turquoise Dhampiralmost 5 years ago

I wasn't doing a screen but a gene specific activation. I had viral particles for dCas9, sgRNA for a specific gene, and MS2, and infected the cells with them simultaneously. As you have correctly pointed out, the triple antibiotics combination was really toxic, together with the variability of only a small population of cells taking up three viral particles, had wiped out all the cells relative to untransduced cells.

I will redo the transduction with only dCas9 and MS2, select for positive cells, before transducing the sgRNAs.

Fuchsia Kelpiealmost 5 years ago

To add to answer, when we do activation we usually make the dCas9/MS2-p65-HSF1 cell line first and then introduce the sgRNA. If your cell line is particularly sensitive to selection, we also have the 2-plasmid lentiSAMv2 system.

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