Use of whole genome CRISPR activation screens to identify genes whose overexpression affects sensitivity to drugs

Authors

• CC Lipsey
• E Ooko
• H Wade
• PJ Batista
• RW Robey
• MM Gottesman

Abstract

The improvement of cancer therapy requires understanding the mechanisms of drug resistance in cancer cells. Studies using genome-wide CRISPR screens have shown promise as a new approach to elucidate resistance mechanisms in various cancer models.

We have conducted CRISPR activation (CRISPRa) screens to explore paclitaxel resistance mechanisms in the cervical cancer cell line KB-3-1 (a HeLa derivative). Cells that stably express dCas 9-VP64 were transduced with the Calabrese small guide RNA (sgRNA) library and treated with paclitaxel (0.1 μM) or in the paclitaxel in the presence of valspodar (1ug/mL) After 14 days of selection, sgRNA distribution was analyzed to determine genes that were overrepresented in paclitaxel-treated cells versus the non-treated control. This screen identified ABCB1(P-glycoprotein, the multidrug efflux pump) as a top hit in the presence of paclitaxel alone. The inhibition of ABCB1, via valspodar, served as a negative regulator of ABCB1 allowing for the identification of additional genes as possible effectors of drug resistance that are not members of the ABC gene family.

This study confirms that the ABC transporter P-glycoprotein is the major mechanism of resistance to paclitaxel in cultured KB-3-1 cells. However, we also show that inhibition of this multidrug efflux pump increases the positive selection of genes that contribute to paclitaxel resistance mechanisms. These data also suggest that coupling CRISPRa with whole-genome screens may provide additional insights into other genes that are major mediators of cancer drug resistance.

Scientific Focus Area: Cancer Biology

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