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

We reported that the integrin αvβ6 is expressed weakly in vivo on lung epithelial cells but its expression is increased during lung damage (John A, J Nucl Med, 2013). Upregulated αvβ6 is implicated in promoting further lung damaging processes via its ability to bind Latency Associated Peptide (LAP) and activate latent-TGFβ (Munger J, Cell, 1999) an extracellular matrix-bound protein. We showed that αvβ6 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 αvβ6 to endocytose on lung epithelia, thus making them unavailable to activate latent-TGFβ, could suppress lung damage during times of lung stress and improve human health. However little is known about the molecular control of αvβ6 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 αvβ6 endocytosis and trafficking. We will generate fluorescently tagged ligands of v6 including a fluorochrome that emits fluorescence at acidic pHs. Using both flow cytometry and immunofluorescence microcopy we will determine optimal concentrations of recombinant ligand for endocytosis in vitro using Normal Human Bronchial Epithelial (NHBE express αvβ6). Double-immunofluorescence labelling for EEA1 (early-endosome, pH variable) and LAMP2 (late endosome, acidic pH) will confirm endocytosed ligands emit fluorescence. To Identify genes that control endocytosis of αvβ6 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. Infected NHBE will be exposed to fluorescent ligands at 4° C for 10’, washed then warmed to 37° C (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 αvβ6 endocytosis. Similarly, the 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 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 treated similalrly and 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 αvβ6 trafficking.