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

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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.