CRISPRi screen to identify genes critical for the shear stress activation of non-adherent monocyte.

Circulating blood cells, such as leukocytes, experience significant hemodynamic stresses. These stresses are significantly higher when a patient requires mechanical support of the circulation, such as cardiopulmonary bypass. To identify molecular mechanisms by which cells sense these stresses, we utilized genome wide CRISPRi and phosphoproteomics data. These screens identified non-erythroid spectrin 1 (SPTAN1) and RAF1 as effectors of hemodynamic stress in monocytes. We show that fluid stress induces SPTAN1/RAF1 signaling to promote STIM1 and ORAI1 interaction resulting in Store-Operated Calcium Entry (SOCE) thereby driving inflammation and cell death. Overall design: IL8-GFP THP-1 cells were transduced with Brunello CRISPRi lentivirus library at multiplicity of infection (MOI) of 1. Cells were sheared in an in vitro system at 2 million cells/mL at a rate of 10mL/min for 2 hours. After shear, cells were prepared for flow cytometry and fluorescence-activated cell sorting (FACS). The 10% brightest GFP cells (LH2) were collected as the experimental group. The next 25% brightest cells (LH3) and unsheared transduced cells were collected. DNA was isolated and sequenced for the gRNAs. MAGeCKFlute was used to analyze the sequencing data.