Fig. 2: Genome-wide CRISPR screen uncovers the expanded network of genes regulating high mannose.

a Schematic for FACS-based CRISPR screen. Cas9-expressing A549s were lentivirally transduced with a genome-wide CRISPR-deletion sgRNA library. Resulting cells were dox-treated to induce XBP1s overexpression for 48 hours. Cells were then gently lifted with Accutase, fixed, and stained with FITC-labeled HHL. The top and bottom 25% of HHL stained cells were isolated by FACS. The resulting populations were subjected to deep sequencing and analysis. The screen was performed in duplicate. b Volcano plot of all genes indicating effect and confidence scores for the genome-wide screen performed in duplicate. Effect and P values were calculated by casTLE. c Schematic for initial steps of N-glycan mannose-trimming and remodeling. All three enzymes indicated are hits in genome-wide screen. d Disruption of tail-anchored protein insertion pathway by ASNA1 inhibitor Retro-2 in wild type A549s also upregulates cell surface high mannose glycan levels. A549s were treated with treated with 2 µg/mL dox, 100 µM of Retro-2, both, or left untreated for 48 hours. Resulting cells were lifted with Accutase and stained with FITC-labeled HHL, followed by flow cytometry analysis. Data are presented as mean ± s.e.m. of median of each replicate and are representative of two independent experiments performed in triplicate with consistent results. p values were calculated from two-tailed Student’s t test. e Schematic for competitive binding assays for measuring changes in high mannose levels. Cells expressing sgRNAs for CRISPRi-mediated knockdown (KD) and miRFP and cells expressing a control sgRNA and BFP were cocultured in 1:1 ratio. Cells were either treated with dox to induce XBP1s or left untreated for 48 hours. Resulting cells were lifted and stained with HHL-FITC, and log2 ratio of HHL intensity of KO: control was determined using flow cytometry. f Validation of hits in XBP1s-induced A549s using competitive HHL binding assays. Data are presented as mean ± s.e.m. and are representative of two independent experiments performed in triplicate with consistent results. p values were calculated from two-tailed Student’s t test.