CRISPR/Cas9 screen to identify mediators of cellular senescence

我们正在研究CRISPR/Cas9筛选技术的可行性，以获得对参与细胞衰老的基因功能的机械性见解，以揭示改进年龄相关疾病临床治疗的新靶点。通过在三种人成纤维细胞系(IMR90、WI-38和BJ)中使用不同细胞衰老触发物(IR、依托泊苷和阿霉素)的浓度范围，我们建立了用衰老相关半乳糖苷酶活性衡量，连续处理50M依托泊苷10天的WI-38细胞获得了最高水平的衰老表型。因此，我们选择了该条件和细胞系作为CRISPR/Cas9筛选的最佳衰老模型。以5500万细胞为转导细胞，以2g/ml的嘌呤霉素为选择剂量，通过实验进一步滴定并优化了筛选条件。为了选择影响衰老细胞活性的基因和识别潜在的衰老靶点，我们将编码Cas9的lentiCRISPRv2(Addgene)文库转导到用依托泊苷诱导衰老的WI-38细胞和作为对照的WI-38增殖细胞中。目前，我们正在准备对来自第一组筛选的细胞中代表的gRNA进行测序。

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.