Cell lines losing Cas9 expression/activity over time - hampering genome-wide screens

We've generated a number of cell lines using Lenti-Cas9-Blast, a mixture of either pooled cell populations if the Cas9 activity was high, or cloned populations if the bulk population had low Cas9 activity (as judged by cell death with sgRNAs targeting known essential genes).

We've found that a fraction of cloned cell lines are losing Cas9 activity during expansion, so that by the time we get enough cells to perform several genome-wide screens simultaneously, cutting efficiency is low, and the screens perform poorly. We're not seeing this in all cell lines though, and I haven't observed this in other cancer cell types.

It is not yet clear to us whether the construct is being silenced, or lost at the genomic level (this cancer type has a lot of genomic instability).

In any case, are others seeing this phenomenon, and how do they deal with it? Switching to another drug marker? It doesn't seem like much else other than fluors for solo Cas9 expression. Perhaps Hygro?

Any thoughts?


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Fuchsia Kelpieover 4 years ago

I have not encountered this issue personally, but typically I would select for blast for 5 days, recover (no antibiotic) for 2 days, and then introduce the sgRNA library. If you need to grow your cells up to have enough cells for screening, and are seeing a loss in Cas9 expression, I would recommend re-selecting for blast right before screening to ensure Cas9 expression.

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