A Perturbation Cell Atlas of KOLF2.1J hiPSCs Reveals Transcriptional and Post-Transcriptional Regulators of Cell State
Abstract
Comprehensively mapping the relationship between genotype and phenotype offers essential insights into how a cell's state arises from its genetic components. Towards this goal, we generated an expressed genome-scale CRISPRi Perturbation Cell Atlas in KOLF2.1J human induced pluripotent stem cells (hiPSCs), mapping transcriptional phenotypes associated with 11,692 perturbed genes across >2.5 million single cells. Using correlations between perturbed phenotypes, we created a cell map of the pluripotent state, demonstrating rich recapitulation of functionally related protein complexes. We then explored the atlas to uncover novel metabolic factors (ZBTB41) and pluripotency regulators (RNF7), validating their functions through metabolic tracing, immunofluorescence, and protein-protein interaction assays. Finally, we leveraged the atlas to generate the first genome-scale screen of RNA editing modulators assayed via direct transcriptome-wide RNA editing, uncovering and mechanistically validating DBR1 as a potent regulator. Taken together, our study presents a comprehensive resource for interrogating the regulatory networks governing pluripotency.
Using the viewer
- MDE
- 2D embedding of the 1,655 strong perturbations, colored by HDBSCAN cluster. Click a point for per-perturbation metadata, D14 fitness, and nearest neighbors.
- Clustermaps
- Pairwise Pearson-r heatmaps of perturbation signatures and of feature-gene z-score profiles. Toggle between Strong perturbations and Feature genes at the top of the tab.
- Gene query
- Pick a feature gene to see which perturbations most strongly up- or down-regulate it, or pick a perturbed gene to see its top up/down transcripts. Runs over all genes and all perturbations passing basic QC (not restricted to the 1,655 strong perturbations). Histogram bars are interactive — hover to see which genes/perts fall in each bin.
- Clusters
- Browse HDBSCAN clusters of strong perturbations with curated labels. Each cluster shows the mean NTC z-score top up / down genes and its member perturbations (click any member to jump to the MDE).
- Gene panel
- Pick up to 10 feature genes (rows) and up to 10 perturbations, or switch the column axis to HDBSCAN clusters, for a pairwise mean NTC z-score heatmap.
Data availability
- Raw FASTQs* : SRA BioProject PRJNA1173491
- Anndata Object (10x processed data file): figshare
*Per usage restrictions from the producers of the KOLF2.1J cell line (Jackson Labs / Genome Research Limited), we are not permitted to openly share this data due to it containing exact read sequences associated with the Y chromosome (see Terms and Conditions). Data is accessible upon emailing the corresponding author with explicit permission from Jackson Labs to share this data. The anndata object contains the counts of reads mapping to the Y chromosome.
Code availability
- Analysis pipeline and Jupyter notebooks: github.com/y-doctor/KOLF2.1J_Perturbation_Cell_Atlas
Citation
Nourreddine, Doctor, et al. A Perturbation Cell Atlas of KOLF2.1J hiPSCs Reveals Transcriptional and Post-Transcriptional Regulators of Cell State. bioRxiv 2024. doi:10.1101/2024.11.03.621734
Pick an expressed feature gene to see the perturbations that most strongly up-regulate or down-regulate it (NTC z-score, post-basic-QC universe of all perturbations).
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Top 50 perturbations · highest z-score
Top 50 perturbations · lowest z-score
Pick a cluster on the left to see its averaged top 50 up/down genes (mean NTC z-score across strong-perturbation members) and the member perts.
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Top 50 genes · highest avg z
Top 50 genes · lowest avg z
Member perturbations (0)
Pick feature genes (rows) and perturbations (cols) to see each pairwise mean NTC z-score. Switch the column axis to HDBSCAN clusters for a cluster-aggregated view.