CRISPR/Cas9 screen to identify mediators of cellular senescence

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We are investigating the feasibility of CRISPR/Cas9 screening technology to gain mechanistic insights into the function of genes involved in cell senescence to reveal novel targets for improved clinical treatments of age-related diseases. By using the concentration range for various triggers of cellular senescence (IR, Etoposide and Doxorubicin) in three human fibroblasts cell lines (IMR90, WI-38 and BJ), we have established that WI-38 cells continuously treated for 10 days with 50 M of Etoposide achieved the highest level of senescent phenotype, as measured by senescence-associated -galactosidase activity. Therefore, we have chosen this condition and cell line as an optimal senescence model for the CRISPR/Cas9 screening. We experimentally further tittered and optimized conditions for the screening as follows using 55 million cells for transduction and 2g/ml puromycin as a selection dose. For selecting genes that affect the viability of senescent cells and for identifying potential senolytic targets, we have transduced the lentiCRISPRv2 (Addgene) library that encodes Cas9 into WI-38 cells after inducing senescence with Etoposide and into WI-38 proliferating cells as a control. Currently, we are in the process of preparation for sequencing of gRNAs represented in the cells from this first set of screening.