Use of CRISPR/Cas9 Genome Library to dissect molecular control of endocytosis and trafficking in lung epithelia

我们报道了整联蛋白α-ν β-6（avb 6）在肺上皮细胞上在体内弱表达，但其表达在肺损伤期间增加（John A，J Nucl Med，2013）。上调的avb 6涉及通过其结合潜伏相关肽（Latency Associated Peptide）和激活潜伏TGF β（Munger J，Cell，1999）（一种细胞外基质结合蛋白）的能力促进进一步的肺损伤过程。我们表明，avb 6整联蛋白经历内吞作用作为其正常生物学功能的一部分（Ramsay A，Cancer Res，2007），但也将内吞和运输非基质结合的配体（Saha A，J Path，2010）。因此，促进avb 6对肺上皮细胞的内吞作用，从而使它们无法激活潜伏性TGF β，可以在肺应激期间抑制肺损伤并改善人类健康。然而，关于正常肺细胞中avb 6内吞作用和细胞内运输的分子调控知之甚少。我们将使用高通量全基因组CRISPR/Cas9基因敲除策略，结合下一代测序来鉴定促进和抑制avb 6内吞和运输的基因。这些结果也可能适用于改善动物健康，因为口蹄疫病毒被avb 6内吞，作为感染牛的一种方式（Berryman，J Virol，2005）。我们将从GSK产生的谷胱甘肽-S-转移酶-GST构建体中产生GFP标记的重组GST（rGST）（Ludbrook SB，Biochem J，2003）。此外，重组GST-GST将与pHrodo（Thermo-Fisher）连接，pHrodo是一种在酸性pH下发射荧光的荧光染料。使用流式细胞术和免疫荧光显微镜，我们将使用正常人支气管上皮细胞（NHBE express avb 6;由GSK提供）确定用于体外内吞作用的重组人β 2（rHBE）的最佳浓度。EEA 1（早期内体，pH可变）和LAMP 2（晚期内体，酸性pH）的双重免疫荧光标记将确认LAP-pHrodo蛋白何时发射荧光。为了鉴定控制avb 6整联蛋白内吞作用的基因，将用慢病毒壁虎lentiCRISPRv 2 Cas9/CRISPR基因组文库（Addgene目录号100000048; Sanjana，N，Nature Methods. 2014年）。将感染的NHBE在4摄氏度下暴露于rLAP-GFP 10 '，洗涤，然后温热至37摄氏度（15'）以允许内吞作用，然后在10 mM HCl中洗涤5'以除去表面结合的配体。使用FACS Aria II细胞分选仪收集非荧光细胞（即内吞作用被抑制）。来自这些细胞的DNA将进行下一代测序（NGS）和生物信息学应用，以识别那些被CRISPR敲除的基因，这些基因通常是促进avb 6内吞作用所需的。类似地，将收集前3%最多荧光的细胞并进行类似分析，因为这些敲除的基因通常抑制内吞作用。生物信息学和NGS培训将提供给博士生在GSK。将鉴定来自NGS分析的最高度富集的靶标，并生成单个CRISPR构建体并将其应用于NHBE细胞，并重复内化实验，以确认关键基因的作用。将粘附的CRISPR/Cas9处理的NHBE暴露于rLAP-pHRodo相似的时间和温度。使用Zeiss荧光PALM激光解剖显微镜收集500个具有荧光核周内体的细胞，提取DNA并进行类似分析以鉴定b6运输的基因。

We reported that the integrin alpha-v beta-6 (avb6) is expressed weakly in vivo on lung epithelial cells but its expression is increased during lung damage (John A, J Nucl Med, 2013). Upregulated avb6 is implicated in promoting further lung damaging processes via its ability to bind Latency Associated Peptide (LAP) and activate latent-TGFbeta (Munger J, Cell, 1999) an extracellular matrix-bound protein. We showed that avb6 integrins undergo endocytosis as part of their normal biological function (Ramsay A, Cancer Res, 2007) but also will endocytose and traffic non-matrix bound ligands (Saha A, J Path, 2010). Thus promoting avb6 to endocytose on lung epithelia, thus making them unavailable to activate latent-TGFbeta, could suppress lung damage during times of lung stress and improve human health. However little is known about the molecular control of avb6 endocytosis and intracellular trafficking in normal lung cells. We shall use a high throughput genome-wide CRISPR/Cas9 gene knockout strategy, combined with next-generation sequencing to identify the genes that both promote and suppress avb6 endocytosis and trafficking. These results are also likely to be applicable to improvement of animal health since foot-and-mouth-disease virus is endocytosed by avb6 as a means of infecting cattle (Berryman, J Virol, 2005). We will Generate GFP-tagged recombinant LAP (rLAP) from Glutathione-S-transferase-LAP constructs generated by GSK (Ludbrook SB, Biochem J, 2003) Additionally, recombinant GST-LAP will be linked to pHrodo (Thermo-Fisher), a fluorochrome that emits fluorescence at acidic pHs. Using both flow cytometry and immunofluorescence microcopy we will determine optimal concentrations of recombinant LAP (rLAP) for endocytosis in vitro using Normal Human Bronchial Epithelial (NHBE express avb6; supplied by GSK). Double-immunofluorescence labelling for EEA1 (early-endosome, pH variable) and LAMP2 (late endosome, acidic pH) will confirm when LAP-pHrodo proteins emit fluorescence. To Identify genes that control endocytosis of avb6 integrins NHBE cells will be transduced (~0.2 viruses per cell to ensure only one gene knockout per cell) with the lentiviral Gecko lentiCRISPRv2 Cas9/CRISPR genome library (Addgene cat#1000000048; Sanjana, N, Nature Methods. 2014). Infected NHBE will be exposed to rLAP-GFP at 4 degrees centigrade for 10', washed then warmed to 37 degrees centigrade (15') to allow endocytosis then washed 5' in 10mM HCl to remove surface bound ligands. Using a FACS Aria II cell sorter non-fluorescent cells (ie endocytosis was inhibited) are collected. DNA from these cells will be subjected to next generation sequencing (NGS) and bioinformatics applied to identify those genes knocked out by CRISPR that normally are required to promote avb6 endocytosis. Similarly, the top 3% most fluorescent cells will be collected and similarly analysed as these knocked-out genes normally suppress endocytosis. Bioinformatics and NGS training will be provided to the PhD student at the GSK. The most highly enriched targets form the NGS analyses will be identified and individual CRISPR constructs generated and applied to NHBE cells and internalization experiments repeated, to confirm the role of key genes. Adherent CRISPR/Cas9 treated NHBE will be exposed to rLAP-pHRodo for similar times and temperatures. 500 cells with fluorescent peri-nuclear endosomes will be collected using a Zeiss fluorescence PALM Laser dissecting microscope, DNA extracted and similarly analysed to identify genes for b6 trafficking.