Journal: bioRxiv

Article Title: Transcriptional regulation of disease-relevant microglial activation programs

doi: 10.1101/2025.10.12.681832

Figure Lengend Snippet: (A) Schematic of FACS-based CRISPRi screens. iPSCs expressing inducible CRISPRi machinery were transduced with a pooled library of sgRNAs targeting ~1600 transcription factors and transcriptional regulators. iPSCs were differentiated into microglia (iTF-MG protocol) and CRISPRi knockdown was induced with trimethoprim. Microglia were stained with markers for six activation states. The 30% of cells with the lowest and highest marker expression were separated by FACS. Genomic DNA was isolated from each population and the cassette encoding sgRNAs was amplified and sequenced using next-generation sequencing. Comparison of the frequencies of cells expressing each sgRNA to those expressing non-targeting control sgRNAs was used to determine hits. (B-G) Volcano plots of screening results for each state. Knockdown phenotype is calculated as a log 2 ratio of counts in the high and low marker expression populations normalized to the standard deviation of non-targeting control cells. CRISPRi targets that significantly (FDR < 0.01) increase or decrease state marker expression are colored in red or blue, respectively. Gene knockdowns that do not significantly alter state marker expression (FDR ≥ 0.01) are shown in grey. Quasi-genes generated from random sets of non-targeting control sgRNAs to model a null distribution are shown in black. (H) Heatmap representing screen results for all genes (rows) that were significant in one or more of the screens shown in H. Each column represents an individual screen. Color scale represents the knockdown phenotype and significance represents FDR. Labelled genes were selected for secondary screens with deep single-cell phenotyping. *FDR < 0.05, **FDR < 0.01, ***FDR < 0.001 FDR.

Article Snippet: For experiments with CRISPRi, cells were supplemented with 50 nM trimethoprim (TMP) (MP Biomedical, 195527) every other day to maintain strong knockdown.

Techniques: Expressing, Transduction, Knockdown, Staining, Activation Assay, Marker, Isolation, Amplification, Next-Generation Sequencing, Comparison, Control, Standard Deviation, Generated