Genome-wide CRISPR screen in human T cells reveals regulators of FOXP3 – Nature

Sakaguchi, S. et al. Regulatory T cells and human disease. Annu. Rev. Immunol. 38, 541–566 (2020).
Google Scholar
Raffin, C., Vo, L. T. & Bluestone, J. A. Treg cell-based therapies: challenges and perspectives. Nat. Rev. Immunol. 20, 158–172 (2020).
Google Scholar
Sakaguchi, S. Taking regulatory T cells into medicine. J. Exp. Med. 218, e20210831 (2021).
Google Scholar
Chen, W. et al. Conversion of peripheral CD4+CD25− naive T cells to CD4+CD25+ regulatory T cells by TGF-β induction of transcription factor Foxp3. J. Exp. Med. 198, 1875–1886 (2003).
Google Scholar
Kanamori, M., Nakatsukasa, H., Okada, M., Lu, Q. & Yoshimura, A. Induced regulatory T cells: their development, stability, and applications. Trends Immunol. 37, 803–811 (2016).
Google Scholar
Floess, S. et al. Epigenetic control of the Foxp3 locus in regulatory T cells. PLoS Biol. 5, e38 (2007).
Google Scholar
Ferreira, L. M. R., Muller, Y. D., Bluestone, J. A. & Tang, Q. Next-generation regulatory T cell therapy. Nat. Rev. Drug Discov. 18, 749–769 (2019).
Google Scholar
Mikami, N., Kawakami, R. & Sakaguchi, S. New Treg cell-based therapies of autoimmune diseases: towards antigen-specific immune suppression. Curr. Opin. Immunol. 67, 36–41 (2020).
Google Scholar
Wei, J. et al. Targeting REGNASE-1 programs long-lived effector T cells for cancer therapy. Nature 576, 471–476 (2019).
Google Scholar
Shifrut, E. et al. Genome-wide CRISPR screens in primary human T cells reveal key regulators of immune function. Cell https://doi.org/10.1016/j.cell.2018.10.024 (2018).
Dong, M. B. et al. Systematic immunotherapy target discovery using genome-scale in vivo CRISPR screens in CD8 T cells. Cell 178, 1189–1204.e23 (2019).
Google Scholar
Cortez, J. T. et al. CRISPR screen in regulatory T cells reveals modulators of Foxp3. Nature 582, 416–420 (2020).
Google Scholar
Loo, C.-S. et al. A genome-wide CRISPR screen reveals a role for the non-canonical nucleosome-remodeling BAF complex in Foxp3 expression and regulatory T cell function. Immunity 53, 143–157.e8 (2020).
Google Scholar
Schumann, K. et al. Functional CRISPR dissection of gene networks controlling human regulatory T cell identity. Nat. Immunol. 21, 1456–1466 (2020).
Google Scholar
Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 34, 184–191 (2016).
Google Scholar
Li, W. et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 15, 554 (2014).
Google Scholar
Tone, Y. et al. Smad3 and NFAT cooperate to induce Foxp3 expression through its enhancer. Nat. Immunol. 9, 194–202 (2007).
Google Scholar
Sun, X., Cui, Y., Feng, H., Liu, H. & Liu, X. TGF-β signaling controls Foxp3 methylation and Treg cell differentiation by modulating Uhrf1 activity. J. Exp. Med. 216, 2819–2837 (2019).
Google Scholar
Hori, S., Nomura, T. & Sakaguchi, S. Control of regulatory T cell development by the transcription factor Foxp3. Science 299, 1057–1061 (2003).
Google Scholar
Seki, A. & Rutz, S. Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells. J. Exp. Med. 215, 985–997 (2018).
Google Scholar
Sauer, M. et al. DHX36 prevents the accumulation of translationally inactive mRNAs with G4-structures in untranslated regions. Nat. Commun. 10, 2421 (2019).
Google Scholar
Zemmour, D. et al. Single-cell gene expression reveals a landscape of regulatory T cell phenotypes shaped by the TCR. Nat. Immunol. 19, 291–301 (2018).
Google Scholar
Luo, Y. et al. Single-cell transcriptomic analysis reveals disparate effector differentiation pathways in human Treg compartment. Nat. Commun. https://doi.org/10.1038/s41467-021-24213-6 (2021).
Chen, K. Y. et al. Joint single-cell measurements of surface proteins, intracellular proteins and gene expression with icCITE-seq. Preprint at bioRxiv https://doi.org/10.1101/2025.01.11.632564 (2025).
Freimer, J. W. et al. Systematic discovery and perturbation of regulatory genes in human T cells reveals the architecture of immune networks. Nat. Genet. 54, 1133–1144 (2022).
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).
Google Scholar
Frangieh, C. J. et al. Multimodal pooled Perturb-CITE-seq screens in patient models define mechanisms of cancer immune evasion. Nat. Genet. 53, 332–341 (2021).
Google Scholar
Araujo, L., Khim, P., Mkhikian, H., Mortales, C.-L. & Demetriou, M. Glycolysis and glutaminolysis cooperatively control T cell function by limiting metabolite supply to N-glycosylation. eLife 6, e21330 (2017).
Google Scholar
Setoguchi, R. et al. Repression of the transcription factor Th-POK by Runx complexes in cytotoxic T cell development. Science 319, 822–825 (2008).
Google Scholar
Wang, L. et al. The zinc finger transcription factor Zbtb7b represses CD8-lineage gene expression in peripheral CD4+ T cells. Immunity 29, 876–887 (2008).
Google Scholar
Oberoi, J. et al. Structural basis for the assembly of the SMRT/NCoR core transcriptional repression machinery. Nat. Struct. Mol. Biol. 18, 177–184 (2011).
Google Scholar
Kao, H. Y. et al. A histone deacetylase corepressor complex regulates the Notch signal transduction pathway. Genes Dev. 12, 2269–2277 (1998).
Google Scholar
Fortini, M. E. & Artavanis-Tsakonas, S. The suppressor of hairless protein participates in Notch receptor signaling. Cell 79, 273–282 (1994).
Google Scholar
Castel, D. et al. Dynamic binding of RBPJ is determined by Notch signaling status. Genes Dev. 27, 1059–1071 (2013).
Google Scholar
Jarriault, S. et al. Signalling downstream of activated mammalian Notch. Nature https://doi.org/10.1038/377355a0 (1995).
Oswald, F. et al. SHARP is a novel component of the Notch/RBP-Jκ signalling pathway. EMBO J. 21, 5417–5426 (2002).
Google Scholar
Oswald, F. et al. A phospho-dependent mechanism involving NCoR and KMT2D controls a permissive chromatin state at Notch target genes. Nucleic Acids Res. 44, 4703–4720 (2016).
Google Scholar
Delacher, M. et al. Rbpj expression in regulatory T cells is critical for restraining T2 responses. Nat. Commun. 10, 1621 (2019).
Google Scholar
Zheng, Y. et al. Role of conserved non-coding DNA elements in the Foxp3 gene in regulatory T-cell fate. Nature 463, 808–812 (2010).
Google Scholar
Feng, Y. et al. Control of the inheritance of regulatory T cell identity by a cis element in the Foxp3 locus. Cell 158, 749–763 (2014).
Google Scholar
Li, X., Liang, Y., LeBlanc, M., Benner, C. & Zheng, Y. Function of a Foxp3 cis-element in protecting regulatory T cell identity. Cell 158, 734–748 (2014).
Google Scholar
Kawakami, R. et al. Distinct Foxp3 enhancer elements coordinate development, maintenance, and function of regulatory T cells. Immunity 54, 947–961.e8 (2021).
Google Scholar
Dikiy, S. et al. A distal Foxp3 enhancer enables interleukin-2 dependent thymic Treg cell lineage commitment for robust immune tolerance. Immunity 54, 931–946.e11 (2021).
Google Scholar
Ohkura, N. et al. T cell receptor stimulation-induced epigenetic changes and Foxp3 expression are independent and complementary events required for Treg cell development. Immunity 37, 785–799 (2012).
Google Scholar
Mikami, N. et al. Epigenetic conversion of conventional T cells into regulatory T cells by CD28 signal deprivation. Proc. Natl Acad. Sci. USA 117, 12258–12268 (2020).
Google Scholar
Yue, X. et al. Control of Foxp3 stability through modulation of TET activity. J. Exp. Med. 213, 377–397 (2016).
Google Scholar
Schmidt, A., Eriksson, M., Shang, M.-M., Weyd, H. & Tegnér, J. Comparative analysis of protocols to induce human CD4+Foxp3+ regulatory T cells by combinations of IL-2, TGF-β, retinoic acid, rapamycin and butyrate. PLoS ONE 11, e0148474 (2016).
Google Scholar
Sher, F. et al. Rational targeting of a NuRD subcomplex guided by comprehensive in situ mutagenesis. Nat. Genet. 51, 1149–1159 (2019).
Google Scholar
Yuan, Z. et al. Structural and functional studies of the RBPJ-SHARP complex reveal a conserved corepressor binding site. Cell Rep. 26, 845–854.e6 (2019).
Heinzel, T. et al. A complex containing N-CoR, mSln3 and histone deacetylase mediates transcriptional repression. Nature 387, 43–48 (1997).
Google Scholar
Mimitou, E. P. et al. Scalable, multimodal profiling of chromatin accessibility, gene expression and protein levels in single cells. Nat. Biotechnol. 39, 1246–1258 (2021).
Google Scholar
Skene, P. J. & Henikoff, S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. eLife 6, e21856 (2017).
Google Scholar
Liu, N. et al. Direct promoter repression by BCL11A controls the fetal to adult hemoglobin switch. Cell 173, 430–442.e17 (2018).
Google Scholar
van der Veeken, J. et al. The transcription factor Foxp3 shapes regulatory T cell identity by tuning the activity of trans-acting intermediaries. Immunity 53, 971–984.e5 (2020).
Google Scholar
Zhou, P. et al. Single-cell CRISPR screens in vivo map T cell fate regulomes in cancer. Nature 624, 154–163 (2023).
Google Scholar
Meyer Zu Horste, G. et al. RBPJ controls development of pathogenic Th17 cells by regulating IL-23 receptor expression. Cell Rep. 16, 392–404 (2016).
Google Scholar
Chen, X. et al. Joint single-cell DNA accessibility and protein epitope profiling reveals environmental regulation of epigenomic heterogeneity. Nat. Commun. 9, 4590 (2018).
Google Scholar
Baskar, R. et al. Integrating transcription-factor abundance with chromatin accessibility in human erythroid lineage commitment. Cell Rep. Methods 2, 100188 (2022).
Google Scholar
Komatsu, N. et al. Heterogeneity of natural Foxp3+ T cells: a committed regulatory T-cell lineage and an uncommitted minor population retaining plasticity. Proc. Natl Acad. Sci. USA 106, 1903–1908 (2009).
Google Scholar
Lee, P. P. et al. A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival. Immunity 15, 763–774 (2001).
Google Scholar
Chiou, S. H. et al. Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing. Genes Dev. 29, 1576–1585 (2015).
Google Scholar
Han, H. et al. Inducible gene knockout of transcription factor recombination signal binding protein-J reveals its essential role in T versus B lineage decision. Int. Immunol. 14, 637–645 (2002).
Google Scholar
National Research Council, Division on Earth and Life Studies, Institute for Laboratory Animal Research & Committee for the Update of the Guide for the Care and Use of Laboratory Animals. Guide for the Care and Use of Laboratory Animals 8th edn (National Academies Press, 2011).
Morita, S., Kojima, T. & Kitamura, T. Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther. 7, 1063–1066 (2000).
Google Scholar
Joung, J. et al. Genome-scale CRISPR–Cas9 knockout and transcriptional activation screening. Nat. Protoc. 12, 828–863 (2017).
Google Scholar
Zhou, Y. et al. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nat. Commun. 10, 1523 (2019).
Google Scholar
Korotkevich, G. et al. Fast gene set enrichment analysis. Preprint at bioRxiv https://doi.org/10.1101/060012 (2021).
Brinkman, E. K. & van Steensel, B. Rapid quantitative evaluation of CRISPR genome editing by TIDE and TIDER. Methods Mol. Biol. 1961, 29–44 (2019).
Google Scholar
Conant, D. et al. Inference of CRISPR edits from Sanger trace data. CRISPR J. 5, 123–130 (2022).
Google Scholar
Schoonenberg, V. A. C. et al. CRISPRO: identification of functional protein coding sequences based on genome editing dense mutagenesis. Genome Biol. 19, 169 (2018).
Google Scholar
Corces, M. R. et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat. Methods 14, 959–962 (2017).
Google Scholar
Buenrostro, J. D., Wu, B., Chang, H. Y. & Greenleaf, W. J. ATAC-seq: a method for assaying chromatin accessibility genome-wide. Curr. Protoc. Mol. Biol. 109, 21.29.1–21.29.9 (2015).
Google Scholar
Akella, N. M., Ciraku, L. & Reginato, M. J. Fueling the fire: emerging role of the hexosamine biosynthetic pathway in cancer. BMC Biol. 17, 52 (2019).
Google Scholar
Xu, C. & Ng, D. T. Glycosylation-directed quality control of protein folding. Nat. Rev. Mol. Cell Biol. 16, 742–752 (2015).
Google Scholar