A genome-scale CRISPRi perturbation atlas of human induced pluripotent stem cells

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 11,739 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.Expressed Genome Wide Perturb-Seq Datasets:1A) (KOLF_Pan_Genome_QC_Filtered.h5ad) The full KOLF2.1J expressed genome-wide Perturb-Seq dataset with basic single QC performed (i.e. dead cell qc; up to Extended Data Fig. 2d). This is found in the file KOLF_Pan_Genome_QC_Filtered. This is the file needed to reproduce all analyses related to the expressed genome wide perturb-seq in the manuscript.1B) (KOLF_Strong_Perturbations.h5ad) The KOLF2.1J expressed genome-wide Perturb-Seq dataset subset to only 1655 Strong Perturbations (plus NTC control). For ease of analysis, all feature genes are included, with the .var column 'Feature_Selected' indicating if that feature gene was selected for downstream analysis.Pilot Screen Datasets:2A) (KOLF_Chromatin_Modifiers_QC_Filtered.h5ad) Pilot Perturb-Seq screen of Chromatin Modifiers, QCed in the same way as the expressed genome scale dataset.2B) (KOLF_Chromatin_Modifiers_Strong_Perturbations.h5ad) Pilot Perturb-Seq screen of Chromatin Modifiers subset to Strong Perturbations3A) (KOLF_Chromatin_Modifiers_QC_Filtered.h5ad) Pilot Perturb-Seq screen of Metabolic Enzymes, QCed in the same way as the expressed genome scale dataset.3B) (KOLF_Chromatin_Modifiers_Strong_Perturbations.h5ad) Pilot Perturb-Seq screen of Metabolic Enzymes subset to Strong Perturbations.FUNDING This work was generously supported by NIH grants (OT2OD032742, R01CA310063, R01HG012351), a CIRM Grant (DISC4-19271), a Department of Defense Grant (W81XWH-22-1-0401), CIRM training grant (EDUC4-12804), UCSD Institutional Funds, and a Rubicon grant from Dutch Research Council (NWO, 019.231EN.013). JNH was supported by an EMBO Postdoctoral Fellowship (ALTF 556-2022). This publication includes data generated at the UC San Diego IGM Genomics Center utilizing an Illumina NovaSeq 6000 that was purchased with funding from a National Institutes of Health SIG grant (#S10 OD026929) National Cancer Institute (P30CA023100). Parts of some figures were illustrated with BioRender.