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. 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.

循环血细胞（如白细胞）会受到显著的血流动力学应力。当患者需要循环机械辅助支持（如心肺转流）时，此类应力水平会显著升高。为阐明细胞感知此类应力的分子机制，本研究使用了全基因组CRISPR干扰（CRISPRi）与磷酸化蛋白质组学数据。上述筛选结果显示，非红细胞血影蛋白1（SPTAN1）与RAF1可作为单核细胞中血流动力学应力的效应分子。本研究证实，流体应力可激活SPTAN1/RAF1信号通路，促进STIM1与ORAI1的相互作用，进而触发钙池操纵性钙内流（SOCE），最终介导炎症反应与细胞死亡。本研究以感染复数（MOI）为1的Brunello CRISPR干扰慢病毒文库转导IL8-GFP标记的THP-1细胞。将细胞置于体外流体剪切系统中，以2×10^6 细胞/mL的密度、10mL/min的流速剪切处理2小时。剪切处理结束后，对细胞进行流式细胞术与荧光激活细胞分选（FACS）制备。收集荧光强度最高的10% GFP阳性细胞（LH2组）作为实验组，同时收集荧光强度次之的25% GFP阳性细胞（LH3组）与未经过剪切处理的转导细胞。提取细胞基因组DNA并对向导RNA（gRNAs）进行测序。使用MAGeCKFlute工具对测序数据进行分析。