medium scale mouse sgrna library  (Addgene inc)

Journal: bioRxiv

Article Title: A genetic screen in enteroendocrine cells reveals mechanisms that control protein sensing and GLP-1 release

doi: 10.64898/2025.11.30.691441

Figure Lengend Snippet: a, Expression levels of CaMPARI and ZIM3-KRAB-dCas9 in each isolated single clones. Clone 2C6 is selected for all the CaMPARI screens and validation shown in this manuscript. b, CRISPRi efficiency by qPCR. Left, Knockdown efficiency for two candidate genes shown in . Right, Knockdown efficiency for all tested target genes. c, FACS screen gating strategy. Top and bottom 35% of CaMPARI photoconversion ratio (red/green) was collected. d, Heatmap showing log/fold change) for each individual sgRNA (5 per gene) targeting the top 50 hits from the tryptone screen. e-g, Phenotype scores for all library genes, comparing FACS screen with interal survival screen control. e, Tryptone screen. f, KCI screen. g, Phenylalanine screen. Pearson correlation coefficient is shown on the plot.

Article Snippet: To perform a pilot screen as outlined in , we first obtained a medium-scale mouse sgRNA library (CRISPRi_v2 sublibrary m1; Addgene #83989).

Techniques: Expressing, Isolation, Clone Assay, Biomarker Discovery, Knockdown, Control

Journal: bioRxiv

Article Title: A genetic screen in enteroendocrine cells reveals mechanisms that control protein sensing and GLP-1 release

doi: 10.64898/2025.11.30.691441

Figure Lengend Snippet: a, Composition of custom sgRNA library for large-scale CRISPRi screening. b-c, Volcano plot and rank plot for custom library screen. b, Significant hits (FDR < 0.05) are highlighted. c, Top 10 hits with highest phenotype scrore [logifold change) x -log 10 {pvalue)] are highlighted. d, Functional protein-protein interaction network for all positive hits by STRING. Line thickness indicates the strength of data support for interaction. Genes with mitochondrial annotation (GO:0005739) are highlighted in red. e, Hit distribution for two most critical mitochondrial energy metabolism pathways, TCA cycle and OXPHOS. Strong hits with FDR< 0.05 are highlighted in black, and weak hits with FDR < 0.1 are labeled in ’gray50’. Non-hit genes with FDR≥ 0.1 are ’gray1O’.

Article Snippet: To perform a pilot screen as outlined in , we first obtained a medium-scale mouse sgRNA library (CRISPRi_v2 sublibrary m1; Addgene #83989).

Techniques: Functional Assay, Labeling

Journal: bioRxiv

Article Title: A genetic screen in enteroendocrine cells reveals mechanisms that control protein sensing and GLP-1 release

doi: 10.64898/2025.11.30.691441

Figure Lengend Snippet: a-c, Valiation of top hits in mitochondrial respiration pathways by CRISPRi KD. a, Schematic for experimental design. b, Integrated calcium activity in STC-1 stably expressing non-targeting control (NTC) or sgRNA targeting top hit genes. c, Relative GLP-1 secretion in STC-1 after CRISPRi KD. d-f, Validation of the role of mitochondrial respiration in amino acid sensing by pharmacological inhibition of OXPHOS Complex I. d, Schematic for experimental design. e, Integrated calcium activity in STC-1 cells pretreated with vehicle or IACS010759. f, Relative GLP-1 secretion in STC-1 after stimulation, with vehicle or IACS010759. g-i, Stimulating OXPHOS boosts EEC activity and GLP-1 secretion. g, Schematic for experimental design. h, Integrat-ed calcium activity in STC-1 stably expressing NTC or sgRNA targeting Luc7I2, an inhibitor of OXPHOS. i, Relative GLP-1 secretion in STC-1 with the indicated perturbation and stimulation. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: To perform a pilot screen as outlined in , we first obtained a medium-scale mouse sgRNA library (CRISPRi_v2 sublibrary m1; Addgene #83989).

Techniques: Activity Assay, Stable Transfection, Expressing, Control, Biomarker Discovery, Inhibition

Journal: bioRxiv

Article Title: A genetic screen in enteroendocrine cells reveals mechanisms that control protein sensing and GLP-1 release

doi: 10.64898/2025.11.30.691441

Figure Lengend Snippet: a-b, Amino acid metabolism and entry into the TCA cycle is required for EEC sensing. a, Gls is a key enzyme required for glutamine metabolism and its entry into TCA cycle, but not for praline or glutamate. b, Integrated calcium activity in STC-1 stably expressing NTC or sgRNA targeting Gls. c-d, Restoring NADH and redox is not sufficient for amino acid sensing when OXPHOS is inhibited. c, Schematic for experimental design. d, Integrated calcium activity in STC-1 with the indicated treatments and stimulation.Cells were pre-treated with vehicle/lACS and/or pyruvate for 1 h before stimulation. e-h, KATPchannel is dispensable for amino acid sensing in STC-1. e, Schematic of KATPchannel, composed of Kcnj11 and Abcc8. f, Gene expression levels of Abcc8 and Kcnj11 in STC-1 vs. NIH3T3 by RNA-seq. g, Rank plot showed neither Abcc8 nor Kcnj11 is a hit from the custom library screen by tryoptone. h, pharmacological inhibition of KATP channel in STC-1 does not increase baseline calcium activity, but moderatly increase acitivty with strong stimulation (5 mg/ml tryptone or KCI). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: To perform a pilot screen as outlined in , we first obtained a medium-scale mouse sgRNA library (CRISPRi_v2 sublibrary m1; Addgene #83989).

Techniques: Activity Assay, Stable Transfection, Expressing, Gene Expression, RNA Sequencing, Inhibition