Project name: Genome-wide CRISPR screen for ER stress-UPR pathway

Description: Perturbation of tissue homeostasis accompanies a diversity of inflammatory pathologies and imposes metabolic constraints at the cellular level that elicit ER stress, protein misfolding, and cell death. In response to ER stress, cells initiate the unfolded protein response (UPR), which determines divergent cell fate decisions, either promoting recovery of ER proteostasis and cell survival or triggering programmed cell death. However, the mechanisms by which the UPR transitions from the adaptive to terminal state are not fully understood. To discover novel UPR pathway regulators that influence the outcome of cellular ER stress responses, we performed genome-scale genetic perturbations. Our genome-wide screens revealed that distinct sets of genes regulate UPR activity and maintenance of ER homeostasis. This expansive dataset provides a rich resource that can be leveraged to elucidate signaling crosstalk during ER stress and discover novel UPR regulators.Overall design: To measure the UPR activity, we generated an endogenous XBP1s-GFP reporter by CRISPR knock-in. On day 0, we transduced the Genome-wide lentiviral library at an MOI of 0.2 into the 40 million cells of HT29 XBP1s-GFP cells, which infected one virus per cell with over 100 coverage of the library. On day 2, we selected the transduced cells by adding puromycin (Invitrogen) at 5 ug/ml. At day 5, we re-plated the cells with fresh media. For the XBP1s-GFP screen, we treated DMSO or 0.25 ug/ml of Tm for 13hrs. We filtered (40 μm) the single cells and added DAPI just before the sort to select the live cell fraction. We sorted the cells based on the GFP intensities using FACS, each of high or low bins containing 15% of the cells. We collected about 15 million cells per bin (a representation of over 200 coverage of the library).There are 3 replicates each for Tm high 15% of bin, Tm low 15% of bin, DMSO high 15% of bin and DMSO low 15% of bin. All samples were sequenced in two sets of paired-end files. Demultiplexing was performed with poolq. Raw data files are not demultiplexed. The file sample_index.csv includes the sample barcodes. The file count.tsv includes the gene barcode.

Data type: Other

Sample scope: Monoisolate

Relevance: Medical

Last updated: 2020-03-11