native lentiCRISPRv2 instead of lentiCRISPRv2 + sgRNA against GFP as a control: bad idea?

In a recent experiment I used lentiviral transduction of CRISPR to knock-out/down a gene of interest in a cell line. As a negative control I used the native lentiCRISPRv2 plasmid (with the 2kb filler) instead of the lentiCRISPRv2 plasmid cloned with a sgRNA against GFP for example. When I added the Puromycin to the cells, many of the cells in the negative control condition died. I know that different types of infectivity issues can explain that, but I just wanted to make sure that the fact that I used the native plasmid with the 2kb filler cannot be responsible for the cell death or the lack of resistance to Puromycin.


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Lavender Succubusabout 4 years ago

I've done the same thing as you and had titers a log lower than when a gRNA was cloned. I think the stuffer does have an impact and including it isn't a great control. Fairly small differences between lenticrispr V1 and V2 change titers by about a log. I ended up targeting a safe harbor locus.

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