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

使用 CRISPR/Cas9 基因组文库剖析肺上皮细胞内吞作用和运输的分子控制

基本信息

批准号：
1906151
负责人：
金额：
--
依托单位：
Queen Mary University of London
依托单位国家：
英国
项目类别：
Studentship
财政年份：
2017
资助国家：
英国
起止时间：
2017 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=studentship-1906151
关键词：
Use CRISPR Cas9 Genome Library

项目摘要

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.

我们报道了整合素α -v β -6 （avb6）在肺上皮细胞体内表达较弱，但在肺损伤期间其表达增加（John A, J nuclear Med, 2013）。上调的avb6通过结合潜伏期相关肽（LAP）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，参与促进进一步的肺损伤过程。我们发现，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），但也会内吞和运输非基质结合配体（Saha A, J Path, 2010）。因此，促进avb6在肺上皮上内吞，从而使它们无法激活潜伏的tgf - β，可以抑制肺应激时的肺损伤，改善人体健康。然而，对于正常肺细胞中avb6胞吞作用和细胞内运输的分子控制知之甚少。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，因为口蹄疫病毒被avb6内吞，作为感染牛的一种手段（Berryman, J Virol, 2005）。我们将从GSK （Ludbrook SB, Biochem J, 2003）生产的谷胱甘肽- s -转移酶LAP构建物中生成gfp标记的重组LAP （rLAP）。此外，重组GST-LAP将与pHrodo （thermofisher）连接，pHrodo是一种荧光染料，在酸性ph下发出荧光。通过流式细胞术和免疫荧光显微复制，我们将确定重组LAP （rLAP）体外内吞作用的最佳浓度，用于正常人支气管上皮细胞（NHBE表达avb6，由GSK提供）。双免疫荧光标记EEA1（早期核内体，pH值可变）和LAMP2（晚期核内体，酸性pH值）将确认LAP-pHrodo蛋白何时发出荧光。为了鉴定控制avb6整合素内吞作用的基因，将使用慢病毒Gecko lentiCRISPRv2 Cas9/CRISPR基因组文库（Addgene cat#1000000048; Sanjana， N, Nature Methods. 2014）转导NHBE细胞（每个细胞约0.2个病毒，以确保每个细胞只有一个基因敲除）。受感染的NHBE将在4摄氏度下暴露于rLAP-GFP中10‘，洗涤后加热到37摄氏度（15’）以允许内吞作用，然后在10mM盐酸中洗涤5'以去除表面结合的配体。使用FACS Aria II细胞分选器收集非荧光细胞（即内吞作用被抑制）。来自这些细胞的DNA将受到下一代测序（NGS）和生物信息学的应用，以鉴定那些被CRISPR敲除的基因，这些基因通常是促进avb6内吞作用所必需的。同样，将收集前3%的荧光细胞并进行类似的分析，因为这些敲除的基因通常会抑制内吞作用。将为GSK的博士生提供生物信息学和NGS培训。将鉴定NGS分析中富集程度最高的靶标，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。粘附的CRISPR/Cas9处理的NHBE将暴露在rLAP-pHRodo中相同的时间和温度。将使用蔡司荧光PALM激光解剖显微镜收集500个具有荧光核周围内体的细胞，提取DNA并进行类似分析以确定b6运输的基因。