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，以获得对细胞衰老相关基因功能的机制性见解，从而揭示改善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（IMR 90、WI-38和BJ）中使用细胞衰老的各种触发物（IR、依托泊苷和阿霉素）的浓度范围，我们已经确定了用50 μ M依托泊苷连续处理10天的WI-38细胞达到了最高水平的衰老表型，如通过衰老相关半乳糖苷酶活性所测量的。因此，我们选择这种条件和细胞系作为CRISPR/Cas9筛选的最佳衰老模型。我们实验性地进一步滴定和优化了筛选条件，如下使用5500万个细胞进行转导和2 μ g/ml嘌呤霉素作为选择剂量。为了选择影响衰老细胞活力的基因和鉴定潜在的衰老清除靶，我们在用依托泊苷诱导衰老后将编码Cas9的lentiCRISPRv 2（Addgene）文库转导到WI-38细胞中，并转导到WI-38增殖细胞中作为对照。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.