Journal: bioRxiv

Article Title: The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC reprogramming

doi: 10.1101/2023.07.31.551297

Figure Lengend Snippet: ( A ) Experimental design. X-GFP iCas9 ESCs were infected with a genome-wide lentiviral gRNA library and differentiated into NPCs. Next, NPCs were treated with doxycycline to activate the expression of the reprogramming cassette and the iCas9 . The knockouts took place during reprogramming. At day 10 of reprogramming, three populations were FACS-separated: non-pluripotent (SSEA1-X-GFP-), early pluripotent (SSEA1+ X-GFP-) and late pluripotent, X-reactivated (SSEA1+ X-GFP+). For these three populations and the NPCs, genomic DNA extraction, PCR-amplification of the gRNA sequences, sequencing and analysis of gRNA abundance were performed. n=2 biological replicates (independent reprogramming rounds). ( B ) Pathways related to overrepresented genes in the three reprogramming populations (non-pluripotent, early pluripotent and late pluripotent) compared to NPCs (WikiPathways Mouse 2019). For pathway enrichment analysis, the top 250 genes from each individual comparison to NPCs ranked by RRA score with MAGeCK software were merged and selected. ( C ) gRNA abundance comparison (early pluripotent vs non-pluripotent) and representation of genes with negative Log2FC (underrepresented) vs −log10 RRA (RRA cutoff = 0.05, Log2FC cutoff = −0.75) (activators of early pluripotency). ( D ) gRNA abundance comparison (late pluripotent vs early pluripotent) and representation of genes with negative Log2FC (underrepresented) vs −log10 RRA (RRA cutoff = 0.05, Log2FC cutoff = −0.75) (activators of late pluripotency, X-reactivation). Genes highlighted in yellow are related to pluripotency, genes highlighted in red are involved in Notch or interferon γ signaling. ( E ) Pathways (WikiPathways Mouse 2019) related to underrepresented genes in “late pluripotent vs early pluripotent” comparison (activators of late pluripotency, X-reactivation, n=1313 genes). Pathways related to proliferation, differentiation and metabolism are shown in gray. The rest of the pathways are highlighted in green. RRA score < 0.05 and Log2FC < −0.8 filtering was applied. ( F ) Experimental design for (G). Treatment with molecules targeting pathways identified in the CRISPR screen was done at the beginning of reprogramming (day 0 - day 5), later in reprogramming (day 5 - day 7) or during the whole process (day 0 - day 7). At day 7, flow cytometry analysis of SSEA1 and X-GFP percentages was performed. ( G ) Pathway validation by molecule treatment at the beginning of reprogramming (day 0 - day 5), later in reprogramming (day 5 - day 7) or during the whole process (day 0 - day 7) in three independent reprogramming rounds, n=3 (except for TGFβ, n=2). Flow cytometry analysis at day 7 of SSEA1 and X-GFP percentages is shown. Data represented as mean +/− SD. Statistics (paired t-tests): where not specified = non-significant; * = p<0.05; ** = p<0.01; *** = p<0.001.

Article Snippet: The gRNA library used for the screening was the Mouse Improved Genome-wide Knockout CRISPR Library v2 (a gift from Kosuke Yusa, Addgene, #67988) , with 90,230 gRNAs targeting 18,424 genes (average of 5 gRNAs per gene).

Techniques: Infection, Genome Wide, Expressing, DNA Extraction, Amplification, Sequencing, Comparison, Software, CRISPR, Flow Cytometry, Biomarker Discovery

Journal: bioRxiv

Article Title: The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC reprogramming

doi: 10.1101/2023.07.31.551297

Figure Lengend Snippet: A genome-wide CRISPR knockout screen reveals molecular networks involved in reprogramming and X-chromosome reactivation. Related to . (A) Validation of knockout efficiency by flow cytometry. Flow cytometry analysis during 6 days of doxycycline treatment in the X-GFP iCas9 ESC line was done to measure the X-GFP percentage decay in cells containing a gRNA targeting the GFP gene. Gating shows the X-GFP+ population. (B) Percentage of gRNA representation in the plasmid library, infected ESCs and the 4 populations analyzed in two independent screening rounds: NPCs and day 10 reprogramming populations (non-pluripotent, early pluripotent, late pluripotent). Error bars represent SD. ( C ) gRNA abundance comparisons (related to D-G): NPCs to non-pluripotent, early pluripotent and late pluripotent populations. ( D ) Pathways related to common underrepresented genes (n=927 genes) in the three reprogramming populations compared to NPCs (WikiPathways Mouse 2019). For all comparisons, a RRA score < 0.05 and Log2FC < −0.75 (underrepresented) filtering was applied. ( E-G ) Representation of genes with negative Log2FC (underrepresented) vs −log10 RRA in the non-pluripotent (E), early pluripotent (F) and late pluripotent (G) populations compared to NPCs (RRA cutoff = 0.05, Log2FC cutoff = −0.75). ( H ) Pathways (WikiPathways Mouse 2019) related to underrepresented genes in the “early pluripotent vs non-pluripotent” comparison (activators of early pluripotency, n=1361 genes) (RRA score < 0.05 and Log2FC < −0.8 filtering was applied). ( I ) Pathways (WikiPathways Mouse 2019) related to overrepresented genes in the “early pluripotent vs non-pluripotent” comparison (repressors of early pluripotency, n=693 genes) (RRA score < 0.05 and Log2FC < −0.8 filtering was applied). ( J ) Representation of genes with positive Log2FC (overrepresented) vs −log10 RRA (RRA cutoff = 0.05, Log2FC cutoff = 0.75) in the “early pluripotent vs non-pluripotent” comparison (repressors of early pluripotency). ( K ) Representation of genes with positive Log2FC (overrepresented) vs −log10 RRA (RRA cutoff = 0.05, Log2FC cutoff = 0.75) in the “late pluripotent vs early pluripotent” comparison (repressors of late pluripotency, X-reactivation). ( L ) Pathways (WikiPathways Mouse 2019) related to overrepresented genes in the “late pluripotent vs early pluripotent” comparison (repressors of late pluripotency, X-reactivation, n=839 genes) (RRA score < 0.05 and Log2FC < −0.8 filtering was applied).

Article Snippet: The gRNA library used for the screening was the Mouse Improved Genome-wide Knockout CRISPR Library v2 (a gift from Kosuke Yusa, Addgene, #67988) , with 90,230 gRNAs targeting 18,424 genes (average of 5 gRNAs per gene).

Techniques: Genome Wide, CRISPR, Knock-Out, Biomarker Discovery, Flow Cytometry, Plasmid Preparation, Infection, Comparison