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

我们报道了整合素α-vβ-6(Avb6)在活体肺上皮细胞上弱表达，但在肺损伤过程中其表达增加(John A，J Nucl Med，2013)。上调的avb6通过结合潜伏期相关肽(LAP)和激活潜伏的TGFbeta(Munger J，Cell，1999)的能力而促进进一步的肺损伤过程。我们表明avb6整合素作为其正常生物学功能的一部分经历内吞作用(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avb6在肺上皮细胞上内吞，从而使其不能激活潜伏的TGFβ，可以抑制肺应激时的肺损伤，改善人类健康。然而，对正常肺细胞中avb6内吞作用和细胞内转运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，结合下一代测序来识别促进和抑制avb6内吞和运输的基因。这些结果也可能适用于改善动物健康，因为口蹄疫病毒是由avb6内吞的，作为感染牛的一种手段(Berryman，J Virol，2005)。我们将从GSK(Ludbrook SB，Biochem J，2003)产生的谷胱甘肽-S-转移酶-LAP结构中产生绿色荧光蛋白标记的重组LAP(RLAP)。此外，重组GST-LAP将连接到pHrodo(Thermo-Fisher)，一种在酸性PHS中发出荧光的荧光色素。使用流式细胞术和免疫荧光显微镜，我们将确定重组LAP(RLAP)的最适浓度，以用于体外利用正常人支气管上皮(NHBE expresse avb6；由GSK提供)进行内吞作用。对EEA1(早期内体，pH可变)和LAMP2(晚期内体，酸性pH)的双重免疫荧光标记将确认LAP-PHRODO蛋白何时发出荧光。为了确定控制AVB6整合素内吞作用的基因，将用慢病毒壁虎慢速CRISPRv2Cas9/CRISPR基因组文库(Addgene CAT#1000000048；Sanjana，N，Natural方法)转导NHBE细胞(每个细胞~0.2个病毒，以确保每个细胞只有一个基因敲除)。2014年)。感染的NHBE将在4摄氏度的RLAP-GFP中暴露10‘，洗涤然后加热到37摄氏度(15’)以允许内吞，然后在10 mM的HCl中洗涤5‘以去除表面结合的配体。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)。这些细胞的DNA将接受下一代测序(NGS)和生物信息学的应用，以识别那些被CRISPR敲除的基因，这些基因通常需要促进avb6内吞。同样，前3%的大多数荧光细胞将被收集并进行类似的分析，因为这些被敲除的基因通常会抑制内吞作用。GSK将为博士生提供生物信息学和NGS培训。将识别来自NGS分析的最高浓缩靶，并生成单个CRISPR结构并将其应用于NHBE细胞和内化实验，以确认关键基因的作用。附着的CRISPR/Cas9处理过的NHBE将在类似的时间和温度下暴露在RLAP-PHRODO中。500个带有荧光核周内体的细胞将使用蔡司荧光掌上激光解剖显微镜收集，提取DNA并进行类似的分析，以确定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.