Journal: bioRxiv

Article Title: A genome wide CRISPR/Cas9 screen identifies calreticulin as a selective repressor of ATF6⍺

doi: 10.1101/2024.02.10.579381

Figure Lengend Snippet: (A) Schematic representation of the strategy for targeted integration at the cgROSA26 safe-harbour locus in the CHO genome, mediated by CRISPR/Cas9-mediated homology-directed repair (HDR). The approach involves an unique sgRNA/Cas9 sequence targeting the cgROSA26 locus and a landing pad donor plasmid containing homology arms (LHA and RHA) flanking a minigene encoding the promoter region of the cgBiP fused with the superfolder green fluorescent protein (sfGFP) to create a BiP::sfGFP reporter to monitor ATF6⍺ activity in cells. (B) Related to , top panel . Quantification of fold-change BiP::sfGFP signals in XC45-6S dual UPR reporter cells under basal conditions (untreated) and treated with the ER stressors 2DG, Tm and Tg (6 h or 20 h). Shown is the normalised (treated/untreated) median fluorescence intensity (MFI) as mean ± SEM of independent repetitions (n > 3). All statistical analysis was performed by a two-sided unpaired Welch’s t-test and significance is indicated by asterisks (**** p < 0.0001). (C) Two-dimensional contour plots of BiP::sfGFP and XBP1s::mCherry levels in XC45-6S cells under basal conditions (UT, grey) or after treatment with tunicamycin alone (2.5 µg/ml, Tm, orange) or in combination with Ceapin A7 (5 µM, green) or S1P inhibitor (15 µM, pink) for 6 h. Histograms of the signal in each channel are displayed on the axes. The bar graph shows the mean ± SEM of the normalised median BiP::sfGFP reporter intensity from more than four independent experiments. All statistical analysis was performed by a two-sided unpaired Welch’s t-test and significance is indicated by asterisks (**** p < 0.0001). (D) Contour plot, as in “S1C”, from parental cells treated with tunicamycin alone (Tm, 2.5 µg/ml, green) or in combination with the IRE1 inhibitor 4µ8C (10 µM, purple) for 18 h. A representative dataset from two independent experiments is shown. (E) Characterisation of the ATF6⍺/IRE1 dual UPR reporter cell line used in the CRISPR/Cas9 screen after stable integration of Cas9. Left panel. Contour plots to confirm Cas9 stable integration. Cells were analysed under basal conditions (grey) or after treatment with 2DG alone (orange) or transfection with an individual lentiGuide-Puro plasmid (without expression of Cas9) targeting exon 18 of IRE1 (encoded by cgERN1 , green). ER stress treatments with 4 mM 2DG lasted 24 h. Right panel . As in “left panel”, but cells were treated with three different ER stressors for 20 h: 2DG (4 mM), Tm (2.5 µg/ml) or Tg (0.5 µM). A representative dataset from two independent experiments is shown.

Article Snippet: For the construction of the wild-type LD repair template (WT; UK3054), a minigene block that contains exons 10 -17 of cgATF6⍺ (785bp; GenScript) was digested with Age I and Afl II and ligated into Age I/ Afl II-digested pUC57 plasmid.

Techniques: CRISPR, Sequencing, Plasmid Preparation, Activity Assay, Fluorescence, Transfection, Expressing