Comprehensive transcription factor perturbations recapitulate fibroblast transcriptional states

comprehensive-transcription-factor-perturbations-recapitulate-fibroblast-transcriptional-states
Comprehensive transcription factor perturbations recapitulate fibroblast transcriptional states
  • Regev, A. et al. The human cell atlas. eLife 6, e27041 (2017).

    PubMed  PubMed Central  Google Scholar 

  • Karlsson, M. et al. A single-cell type transcriptomics map of human tissues. Sci. Adv. 7, eabh2169 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Consortium, T. T. S. et al. The tabula sapiens: a multiple-organ, single-cell transcriptomic atlas of humans. Science 376, eabl4896 (2022).

    Google Scholar 

  • Jain, S. et al. Advances and prospects for the Human BioMolecular Atlas Program (HuBMAP). Nat. Cell Biol. 25, 1089–1100 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Frenkel, M. & Raman, S. Discovering mechanisms of human genetic variation and controlling cell states at scale. Trends Genet. 40, 587–600 (2024).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Rafelski, S. M. & Theriot, J. A. Establishing a conceptual framework for holistic cell states and state transitions. Cell 187, 2633–2651 (2024).

    CAS  PubMed  Google Scholar 

  • Zhang, M. et al. Molecularly defined and spatially resolved cell atlas of the whole mouse brain. Nature 624, 343–354 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Patel, A. S. & Yanai, I. A developmental constraint model of cancer cell states and tumor heterogeneity. Cell 187, 2907–2918 (2024).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Rood, J. E., Maartens, A., Hupalowska, A., Teichmann, S. A. & Regev, A. Impact of the Human Cell Atlas on medicine. Nat. Med. 28, 2486–2496 (2022).

    CAS  PubMed  Google Scholar 

  • Joung, J. et al. A transcription factor atlas of directed differentiation. Cell 186, 209–229.e26 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Gilbert, L. A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR–Cas9 complex. Nature 517, 583–588 (2015).

    CAS  PubMed  Google Scholar 

  • Liu, Y. et al. CRISPR activation screens systematically identify factors that drive neuronal fate and reprogramming. Cell Stem Cell 23, 758–771.e8 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Norman, T. M. et al. Exploring genetic interaction manifolds constructed from rich single-cell phenotypes. Science 365, 786–793 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Tian, R. et al. Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis. Nat. Neurosci. 24, 1020–1034 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Yang, J. et al. Genome-scale CRISPRa screen identifies novel factors for cellular reprogramming. Stem Cell Rep. 12, 757–771 (2019).

    CAS  Google Scholar 

  • Sanson, K. R. et al. Optimized libraries for CRISPR–Cas9 genetic screens with multiple modalities. Nat. Commun. 9, 5416 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Papalexi, E. et al. Characterizing the molecular regulation of inhibitory immune checkpoints with multimodal single-cell screens. Nat. Genet. 53, 322–331 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Griffith, A. L. et al. Optimization of Cas12a for multiplexed genome-scale transcriptional activation. Cell Genomics 3, 100387 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Adamson, B. et al. A multiplexed single-cell CRISPR screening platform enables systematic dissection of the unfolded protein response. Cell 167, 1867–1882.e21 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Dixit, A. et al. Perturb-seq: dissecting molecular circuits with scalable single-cell RNA profiling of pooled genetic screens. Cell 167, 1853–1866.e17 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Bock, C. et al. High-content CRISPR screening. Nat. Rev. Methods Prim. 2, 8 (2022).

    CAS  Google Scholar 

  • Pacalin, N. M. et al. Bidirectional epigenetic editing reveals hierarchies in gene regulation. Nat. Biotechnol. 43, 355–368 (2025).

    CAS  PubMed  Google Scholar 

  • Schmidt, R. et al. CRISPR activation and interference screens decode stimulation responses in primary human T cells. Science 375, eabj4008 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Chardon, F. M. et al. Multiplex, single-cell CRISPRa screening for cell type specific regulatory elements. Nat. Commun. 15, 8209 (2024).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Replogle, J. M. et al. Mapping information-rich genotype–phenotype landscapes with genome-scale Perturb-seq. Cell 185, 2559–2575.e28 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Smith, M. H. et al. Drivers of heterogeneity in synovial fibroblasts in rheumatoid arthritis. Nat. Immunol. 24, 1200–1210 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wei, K. et al. Notch signalling drives synovial fibroblast identity and arthritis pathology. Nature 582, 259–264 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Mizoguchi, F. et al. Functionally distinct disease-associated fibroblast subsets in rheumatoid arthritis. Nat. Commun. 9, 789 (2018).

    PubMed  PubMed Central  Google Scholar 

  • Smillie, C. S. et al. Intra- and Inter-cellular rewiring of the human colon during ulcerative colitis. Cell 178, 714–730.e22 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kinchen, J. et al. Structural remodeling of the human colonic mesenchyme in inflammatory bowel disease. Cell 175, 372–386.e17 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Cadinu, P. et al. Charting the cellular biogeography in colitis reveals fibroblast trajectories and coordinated spatial remodeling. Cell 187, 2010–2028.e30 (2024).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Tsukui, T., Wolters, P. J. & Sheppard, D. Alveolar fibroblast lineage orchestrates lung inflammation and fibrosis. Nature 631, 627–634 (2024).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Tsukui, T. et al. Collagen-producing lung cell atlas identifies multiple subsets with distinct localization and relevance to fibrosis. Nat. Commun. 11, 1920 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Amrute, J. M. et al. Targeting immune–fibroblast cell communication in heart failure. Nature 635, 423–433 (2024).

    CAS  PubMed  Google Scholar 

  • Alexanian, M. et al. Chromatin remodelling drives immune cell–fibroblast communication in heart failure. Nature 635, 434–443 (2024).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Chen, X. & Song, E. Turning foes to friends: targeting cancer-associated fibroblasts. Nat. Rev. Drug Discov. 18, 99–115 (2019).

    CAS  PubMed  Google Scholar 

  • Pradhan, R. N., Krishnamurty, A. T., Fletcher, A. L., Turley, S. J. & Müller, S. A bird’s eye view of fibroblast heterogeneity: a pan-disease, pan-cancer perspective. Immunological Rev. 302, 299–320 (2021).

    CAS  Google Scholar 

  • Buechler, M. B. et al. Cross-tissue organization of the fibroblast lineage. Nature 593, 575–579 (2021).

    CAS  PubMed  Google Scholar 

  • Korsunsky, I. et al. Cross-tissue, single-cell stromal atlas identifies shared pathological fibroblast phenotypes in four chronic inflammatory diseases. Med 3, 481–518.e14 (2022).

    CAS  PubMed  Google Scholar 

  • Gao, Y. et al. Cross-tissue human fibroblast atlas reveals myofibroblast subtypes with distinct roles in immune modulation. Cancer Cell 42, 1764–1783.e10 (2024).

    CAS  PubMed  Google Scholar 

  • Croft, A. P. et al. Distinct fibroblast subsets drive inflammation and damage in arthritis. Nature 570, 246–251 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Krishnamurty, A. T. et al. LRRC15+ myofibroblasts dictate the stromal setpoint to suppress tumour immunity. Nature 611, 148–154 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  • McCartney, E. E., Chung, Y. & Buechler, M. B. Life of Pi: exploring functions of Pi16+ fibroblasts. F1000Res 13, 126 (2024).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Melms, J. C. et al. A molecular single-cell lung atlas of lethal COVID-19. Nature 595, 114–119 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Horlbeck, M. A. et al. Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. eLife 5, e19760 (2016).

    PubMed  PubMed Central  Google Scholar 

  • Replogle, J. M. et al. Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors. eLife 11, e81856 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Gasperini, M. et al. A genome-wide framework for mapping gene regulation via cellular genetic screens. Cell 176, 377–390.e19 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Yao, D. et al. Scalable genetic screening for regulatory circuits using compressed Perturb-seq. Nat. Biotechnol. 42, 1282–1295 (2024).

    CAS  PubMed  Google Scholar 

  • Morris, J. A. et al. Discovery of target genes and pathways at GWAS loci by pooled single-cell CRISPR screens. Science 380, eadh7699 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Datlinger, P. et al. Ultra-high-throughput single-cell RNA sequencing and perturbation screening with combinatorial fluidic indexing. Nat. Methods 18, 635–642 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wu, B. et al. Overloading and unpacKing (OAK)—droplet-based combinatorial indexing for ultra-high throughput single-cell multiomic profiling. Nat. Commun. 15, 9146 (2024).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wu, Q. et al. Massively parallel characterization of CRISPR activator efficacy in human induced pluripotent stem cells and neurons. Mol. Cell 83, 1125–1139.e8 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Loyfer, N. et al. A DNA methylation atlas of normal human cell types. Nature 613, 355–364 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Cazares, T. A. et al. maxATAC: genome-scale transcription-factor binding prediction from ATAC-seq with deep neural networks. PLoS Comput. Biol. 19, e1010863 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Tribolet-Hardy, J. et al. Genetic features and genomic targets of human KRAB-zinc finger proteins. Genome Res. 33, 1409–1423 (2023).

    PubMed  PubMed Central  Google Scholar 

  • O’Geen, H., Henry, I. M., Bhakta, M. S., Meckler, J. F. & Segal, D. J. A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture. Nucleic Acids Res. 43, 3389–3404 (2015).

    PubMed  PubMed Central  Google Scholar 

  • Rostain, W. et al. Cas9 off-target binding to the promoter of bacterial genes leads to silencing and toxicity. Nucleic Acids Res. 51, 3485–3496 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Uthaya et al. A genome-wide CRISPR activation screen identifies SCREEM a novel SNAI1 super-enhancer demarcated by eRNAs. Front. Mol. Biosci. 10, 1110445 (2023).

    Google Scholar 

  • Nuñez, J. K. et al. Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing. Cell 184, 2503–2519.e17 (2021).

    PubMed  PubMed Central  Google Scholar 

  • Li, Y. et al. Genome-wide analyses reveal a role of Polycomb in promoting hypomethylation of DNA methylation valleys. Genome Biol. 19, 18 (2018).

    PubMed  PubMed Central  Google Scholar 

  • Cui, A. et al. Dictionary of immune responses to cytokines at single-cell resolution. Nature 625, 377–384 (2024).

    CAS  PubMed  Google Scholar 

  • Fang, F. et al. Early growth response 3 (Egr-3) is induced by transforming growth factor-β and regulates fibrogenic responses. Am. J. Pathol. 183, 1197–1208 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Li, A. et al. GATA6 triggers fibroblast activation and tracheal fibrosis through the Wnt/β-catenin pathway. Cell. Signal. 105, 110593 (2023).

    CAS  PubMed  Google Scholar 

  • Stebler, S. & Raghunath, M. The Scar-in-a-Jar: In Vitro Fibrosis Model for Anti-Fibrotic Drug Testing. In Myofibroblasts: Methods and Protocols (eds. Hinz, B. & Lagares, D.) 147–156 (Springer, 2021). https://doi.org/10.1007/978-1-0716-1382-5_11

  • Vázquez-García, I. et al. Ovarian cancer mutational processes drive site-specific immune evasion. Nature 612, 778–786 (2022).

    PubMed  PubMed Central  Google Scholar 

  • Pollak, N. M., Hoffman, M., Goldberg, I. J. & Drosatos, K. Krüppel-like factors: crippling and uncrippling metabolic pathways. JACC Basic Transl. Sci. 3, 132–156 (2018).

    PubMed  PubMed Central  Google Scholar 

  • Varrault, A. et al. Identification of Plagl1/Zac1 binding sites and target genes establishes its role in the regulation of extracellular matrix genes and the imprinted gene network. Nucleic Acids Res. 45, 10466–10480 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Jonsson, M. K. B. et al. A transcriptomic and epigenomic comparison of fetal and adult human cardiac fibroblasts reveals novel key transcription factors in adult cardiac fibroblasts. JACC Basic Transl. Sci. 1, 590–602 (2016).

    PubMed  PubMed Central  Google Scholar 

  • Tsuda, T. et al. Zinc finger protein Zac1 is expressed in chondrogenic sites of the mouse. Dev. Dyn. 229, 340–348 (2004).

    CAS  PubMed  Google Scholar 

  • Chrysanthopoulou, A. et al. Down-regulation of KLF2 in lung fibroblasts is linked with COVID-19 immunofibrosis and restored by combined inhibition of NETs, JAK-1/2 and IL-6 signaling. Clin. Immunol. 247, 109240 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Shi, J. et al. KLF2 attenuates bleomycin-induced pulmonary fibrosis and inflammation with regulation of AP-1. Biochem. Biophys. Res. Commun. 495, 20–26 (2018).

    CAS  PubMed  Google Scholar 

  • Chandran, R. R. et al. Distinct roles of KLF4 in mesenchymal cell subtypes during lung fibrogenesis. Nat. Commun. 12, 7179 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Penke, L. R. et al. KLF4 is a therapeutically tractable brake on fibroblast activation that promotes resolution of pulmonary fibrosis. JCI Insight 7, e160688 (2022).

    PubMed  PubMed Central  Google Scholar 

  • Noda, S. et al. Simultaneous downregulation of KLF5 and Fli1 is a key feature underlying systemic sclerosis. Nat. Commun. 5, 5797 (2014).

    CAS  PubMed  Google Scholar 

  • Roadmap Epigenomics Consortium et al. Integrative analysis of 111 reference human epigenomes. Nature 518, 317–330 (2015).

    PubMed Central  Google Scholar 

  • Wang, X. et al. Antibody-free profiling of transcription factor occupancy during early embryogenesis by FitCUT&RUN. Genome Res. 32, 378–388 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  • Southard, K. et al. Hs27-CRISPRa-TFs cellranger outputs. Zenodo https://doi.org/10.5281/ZENODO.15213597 (2025).

  • Southard, K. et al. Hs27-CRISPRa-TFs. Zenodo https://doi.org/10.5281/ZENODO.15200179 (2025).

  • Southard, K. et al. RPE1-CRISPRa-TFs cellranger outputs. Zenodo https://doi.org/10.5281/ZENODO.15211972 (2025).

  • Southard, K. et al. RPE1-CRISPRa-TFs. Zenodo https://doi.org/10.5281/ZENODO.15213619 (2025).

  • Southard, K. et al. RPE1-E150-Benchmarking cellranger outputs. Zenodo https://doi.org/10.5281/ZENODO.15215389 (2025).

  • Southard, K. et al. RPE1-E150-Benchmarking. Zenodo https://doi.org/10.5281/ZENODO.15215414 (2025).

  • Southard, K. et al. K562 dCas9-CUT&RUN. Zenodo https://doi.org/10.5281/ZENODO.15215154 (2025).

  • Southard, K. et al. RPE-1 and Hs27 CUT&RUN, ATAC-seq, and RNA-seq characterization. Zenodo https://doi.org/10.5281/ZENODO.15215216 (2025).

  • K. Southard & Norman, T. norman-lab-msk/TFs_CRISPRa: CRISPRa TFs Perturb-seq v0.1. Zenodo https://doi.org/10.5281/ZENODO.15373940 (2025).