Abstract

Cellular senescence is enhanced in a number of chronic diseases. Several studies have now linked senescent cells to chronic disease pathology and have shown that inhibition/removal of these cells ameliorates disease in relevant models. Senescent cells undergo a form of cell cycle arrest that results in transcriptional and metabolic changes that enhances cellular morphology, cell cycle arrest markers such as p21 and p16 and produce a Senescence Associated Secretory Phenotype (SASP) which includes secretion of cytokines, chemokines and proteases that are thought to contribute to disease pathogenesis. One such debilitating disease is Chronic Obstructive Pulmonary Disease (COPD), where senescent cells are increased and believed to contribute to the disease. In order to identify modulators of cellular senescence for the treatment of COPD we designed an arrayed CRISPRn screen in primary small airway epithelial cells where we screened 250 genes to assess their ability to modulate markers of senescence. For this, we developed a 384-well plate based, semi-automated electroporation workflow to edit these cells using Cas9 RNP which resulted in a high editing efficiency (>90%). 72h following electroporation the cells were treated with Etoposide for 48h to induce senescence or DMSO as a control to measure baseline senescence levels. Careful culture conditions and assay optimisation were required to prevent these cells from senescing naturally to obtain a good assay window for measuring etoposide-induced senescence. This immunofluorescence assay format enabled multiparametric readouts including nuclear p21, enlarged morphology, cell number, cell cycle and phospho-yH2AX foci. These endpoints not only prioritised targets for their ability to modulate senescence, but also informed on the possible mechanisms and safety risks associated with the targets. In addition, the ability to get several informative endpoints from a single assay aligns with our commitment to work more sustainably within functional genomics at AstraZeneca.